TWI700367B - Genetically modified microorganism and method for producing indigo dye - Google Patents
Genetically modified microorganism and method for producing indigo dye Download PDFInfo
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本發明係關於靛藍染料(indigo dye)之生產，且特別關於用於生產靛藍染料之經基因改質的微生物及其製備方法、生產靛藍染料的方法與培養基用於培養經基因改質的微生物並使其生產靛藍染料的用途。The present invention relates to the production of indigo dye (indigo dye), and in particular to a genetically modified microorganism used for the production of indigo dye and a preparation method thereof, a method for producing indigo dye and a medium for cultivating genetically modified microorganisms and Use it to produce indigo dye.
靛藍染料可藉由化學法與生物法生產。於現有靛藍染料之化學法生產製程中，所產生之中間物包括甲醛以及氰化氫，其具有高度污染性。生物法生產靛藍染料是透過微生物醱酵將前驅物(precursor)，例如是色胺酸(tryptophan)或是吲哚(indole)，經由氧化酵素轉化成靛藍染料，然而，生物法生產靛藍目前的產量仍難進入工業化生產規模。Indigo dye can be produced by chemical and biological methods. In the current chemical production process of indigo dye, the intermediates produced include formaldehyde and hydrogen cyanide, which are highly polluting. The production of indigo dye by biological method uses microbial fermentation to convert precursors, such as tryptophan or indole, into indigo dye through oxidative enzymes. However, the current production of indigo by biological methods It is still difficult to enter the scale of industrial production.
麩胺酸(glutamic acid, GA)為廣泛使用之調味料，售價低且料源充足，但目前尚無可有效利用麩胺酸為原料以生產靛藍染料的菌株與生產方式。Gluamic acid (GA) is a widely used seasoning with low price and sufficient raw materials. However, there is no strain and production method that can effectively use glutamic acid as a raw material to produce indigo dye.
因此，目前亟需建立一種以麩胺酸為原料，且利用生物法生產靛藍之技術，以降低靛藍的生產成本。Therefore, there is an urgent need to establish a technology for producing indigo using glutamic acid as a raw material and using a biological method to reduce the production cost of indigo.
本發明提供一種經基因改質的微生物，包括：外源性核酸序列，其編碼萘二氧酶(naphthalene dioxygenase, NDO)，其中上述經基因改質的微生物之內源性 icd基因被剔除，該內源性 icd基因編碼異檸檬酸脫氫酶(isocitrate dehydrogenase, IDH)。上述經基因改質的微生物具有利用麩胺酸及/或其鹽類作為氮源以進行生長並生產靛藍染料(indigo)之能力。 The present invention provides a genetically modified microorganism, comprising: an exogenous nucleic acid sequence encoding naphthalene dioxygenase (NDO), wherein the endogenous icd gene of the above-mentioned genetically modified microorganism is deleted, the The endogenous icd gene encodes isocitrate dehydrogenase (IDH). The above-mentioned genetically modified microorganisms have the ability to use glutamic acid and/or its salts as a nitrogen source to grow and produce indigo.
本發明也提供一種新穎之大腸桿菌基因改質株，其寄存編號為BCRC 940687。上述新穎之大腸桿菌基因改質株之內源性 icd基因被剔除，且包括編碼萘二氧酶之外源性核酸序列。 The present invention also provides a novel genetically modified strain of Escherichia coli, whose deposit number is BCRC 940687. The endogenous icd gene of the novel E. coli genetically modified strain has been deleted, and it includes an exogenous nucleic acid sequence encoding naphthalene dioxygenase.
本發明另提供一種製備經基因改質的微生物的方法，包括以下步驟：(a) 將一微生物之內源性 icd基因剔除以獲得一經 icd基因剔除的微生物；(b) 以含有麩胺酸及/或其鹽類的培養基對該經icd基因剔除的微生物進行一馴化，以獲得一經馴化之微生物；以及(c) 將編碼萘二氧酶之一外源性核酸序列導入該經馴化之微生物，以獲得該經基因改質的微生物。 The present invention also provides a method for preparing a genetically modified microorganism, which includes the following steps: (a) removing the endogenous icd gene of a microorganism to obtain an icd gene-eliminated microorganism; (b) containing glutamine and / Or its salt culture medium for domestication of the microorganism that has been knocked out of the icd gene to obtain a domesticated microorganism; and (c) introducing an exogenous nucleic acid sequence encoding naphthalene dioxygenase into the domesticated microorganism, To obtain the genetically modified microorganism.
本發明還提供一種生產靛藍染料的方法，包括以下步驟：(a) 提供一經基因改質之微生物；以及(b) 將該經基因改質之微生物培養於含有一麩胺酸及/或其鹽類與一靛藍染料前驅物之一培養基中，以產生靛藍染料。該經基因改質之微生物包括上述之經基因改質的微生物、上述之新穎之大腸桿菌基因改質株、或藉由上述之製備經基因改質的微生物的方法所製備的經基因改質的微生物。The present invention also provides a method for producing indigo dye, including the following steps: (a) providing a genetically modified microorganism; and (b) cultivating the genetically modified microorganism on a glutamic acid and/or its salt Class with an indigo dye precursor in a medium to produce indigo dye. The genetically modified microorganisms include the above-mentioned genetically modified microorganisms, the above-mentioned novel E. coli genetically modified strains, or the above-mentioned genetically modified microorganisms prepared by the method for preparing genetically modified microorganisms microorganism.
本發明更提供一種培養基用於培養一經基因改質的微生物並使其生產靛藍染料的用途，其中該培養基包括麩胺酸及/或其鹽類，且於該培養基中，該麩胺酸及/或其鹽類的濃度為0.05-80 g/L，又其中該經基因改質的微生物包括上述外源性核酸序列，其編碼萘二氧酶。The present invention further provides a medium for culturing a genetically modified microorganism to produce indigo dye, wherein the medium includes glutamic acid and/or its salts, and in the medium, the glutamic acid and/ The concentration of the salt thereof is 0.05-80 g/L, and the genetically modified microorganism includes the aforementioned exogenous nucleic acid sequence, which encodes a naphthalene dioxygenase.
為了讓本發明之上述和其他目的、特徵、和優點能更明顯易懂，下文特舉較佳實施例，並配合所附圖示，作詳細說明如下：In order to make the above and other objects, features, and advantages of the present invention more obvious and understandable, the following is a detailed description of the preferred embodiments together with the accompanying drawings:
本發明提供一種經基因改質的微生物。本發明之經基因改質的微生物係可用於生產靛藍染料。The invention provides a genetically modified microorganism. The genetically modified microorganism of the present invention can be used to produce indigo dye.
本發明之經基因改質的微生物具有利用麩胺酸及/或其鹽類作為氮源以進行生長並生產靛藍染料(indigo)之能力。由於麩胺酸售價低且料源充足，因此藉由可高效率利用麩胺酸及/或其鹽類之本發明之經基因改質的微生物，可有效降低靛藍染料之生產成本。The genetically modified microorganism of the present invention has the ability to use glutamic acid and/or its salt as a nitrogen source to grow and produce indigo. Due to the low price of glutamic acid and sufficient raw materials, the genetically modified microorganism of the present invention, which can efficiently utilize glutamic acid and/or its salts, can effectively reduce the production cost of indigo dye.
上述本發明之經基因改質的微生物之微生物來源可包括，但不限於細菌或真菌。The source of the above-mentioned genetically modified microorganism of the present invention may include, but is not limited to, bacteria or fungi.
本發明之經基因改質的微生物之微生物來源之細菌的例子，可包括，艾氏菌屬( Escherichia)、腸桿菌屬( Enterobacter)、腸球菌屬( Enterococcus)、乳酸桿菌屬( Lactobacillus)、乳球菌屬( Lactococcus)、假單孢菌屬( Pseudomonas)、檸檬酸桿菌屬( Citrobacter)、棒狀桿菌屬( Corynebacterium)、伊文氏桿菌屬( Erwinia)、克雷白氏菌屬( Klebsiella)、摩根氏菌屬( Morganella)、泛菌屬( Pantoea)、果膠桿菌屬( Pectobacterium)、變形桿菌屬( Proteus)、沙門桿菌屬( Salmonella)、鋸桿菌屬( Serratia)、志賀桿菌屬( Shigella)等之細菌，但不限於此。而艾氏菌屬之細菌的例子可包括大腸桿菌( Escherichia coli)等，但不限於此。又，上述大腸桿菌可包括大腸桿菌BW25113、K12、DH5a、BL21、或XL1-blue，但不限於此。 Examples of microorganisms of the bacterial origin of genetically modified microorganisms of the present invention, may comprise, Ai Escherichia (Escherichia), Enterobacter (Enterobacter), Enterococcus (of Enterococcus), Lactobacillus (Lactobacillus), milk Lactococcus ( Lactococcus ), Pseudomonas ( Pseudomonas ), Citrobacter ( Citrobacter ), Corynebacterium ( Corynebacterium ), Evans ( Erwinia ), Klebsiella ( Klebsiella ), Morgan genus (Morganella), Pantoea (Pantoea), pectin genus (Pectobacterium), Proteus (Proteus), the genus Salmonella (Salmonella), saw genus (Serratia), the genus Shigella (Shigella), etc. The bacteria, but not limited to this. Examples of bacteria belonging to the genus Escherichia include Escherichia coli , but are not limited thereto. In addition, the above-mentioned Escherichia coli may include Escherichia coli BW25113, K12, DH5a, BL21, or XL1-blue, but is not limited thereto.
而，上述本發明之經基因改質的微生物之微生物來源之真菌的例子，則可包括，耶氏酵母菌屬( Yarrowia)、畢赤酵母菌屬( Pichia)、、紅酵母屬( Rhodotorula)、酵母菌屬( Saccharomyces)、德克酵母屬( Dekkera)、麴菌屬( Aspergillus)、克魯維酵母屬( Kluyveromyces)、青黴菌屬( Penicillium)、黑穗病菌屬( Ustilago)等之真菌，但也不限於此。 However, the above-mentioned examples of the fungi derived from the microorganisms of the genetically modified microorganisms of the present invention may include Yarrowia , Pichia , Rhodotorula , Saccharomyces (Saccharomyces), the genus Dekkera (Dekkera), the genus aspergillus (Aspergillus), Kluyveromyces (Kluyveromyces), Penicillium (Penicillium), the genus smut (Ustilago), etc. fungi, but Not limited to this.
在一實施例中，本發明之經基因改質的微生物之微生物來源可為大腸桿菌，例如大腸桿菌BW25113。In one embodiment, the microorganism source of the genetically modified microorganism of the present invention may be Escherichia coli, such as Escherichia coli BW25113.
在一實施例中，本發明之經基因改質的微生物，可包括編碼萘二氧酶(naphthalene dioxygenase, NDO)之一外源性核酸序列，但不限於此。In one embodiment, the genetically modified microorganism of the present invention may include an exogenous nucleic acid sequence encoding naphthalene dioxygenase (NDO), but is not limited thereto.
上述編碼萘二氧酶之外源性核酸序列可為任何序列，只要其所編碼出之蛋白質具有萘二氧酶之功效即可。例如，上述編碼萘二氧酶之外源性核酸序列可包括與序列辨識號：1之序列具有至少85%之序列相似度的一序列，但不限於此。在一實施例中，上述編碼萘二氧酶之外源性核酸序列可包括序列辨識號：1之序列。在另一實施例中，上述編碼萘二氧酶之外源性核酸序列可為序列辨識號：1之序列。The aforementioned exogenous nucleic acid sequence encoding naphthalene dioxygenase can be any sequence, as long as the protein encoded by it has the effect of naphthalene dioxygenase. For example, the aforementioned exogenous nucleic acid sequence encoding naphthalene dioxygenase may include a sequence with at least 85% sequence similarity to the sequence of sequence identification number: 1, but is not limited thereto. In one embodiment, the above-mentioned exogenous nucleic acid sequence encoding naphthalene dioxygenase may include a sequence of sequence identification number: 1. In another embodiment, the above-mentioned exogenous nucleic acid sequence encoding naphthalene dioxygenase may be the sequence of sequence identification number: 1.
又， icd基因為編碼在三羧酸循環(tricarboxylic acid cycle, TCA)中之異檸檬酸脫氫酶(isocitrate dehydrogenase, IDH)的基因。若於三羧酸循環中，異檸檬酸脫氫酶無法發揮效用，則有較多之麩胺酸可進入氮循環(nitrogen cycle)中，而藉此促進微生物將麩胺酸作為氮源利用的效率。因此，本發明之經基因改質的微生物除了可包括編碼萘二氧酶之外源性核酸序列以外，其內源性 icd基因也可進一步被剔除，以進一步促進微生物對麩胺酸的利用效率。 In addition, the icd gene is a gene encoding isocitrate dehydrogenase (IDH) in the tricarboxylic acid cycle (TCA). If isocitrate dehydrogenase is not effective in the tricarboxylic acid cycle, more glutamate can enter the nitrogen cycle, thereby promoting microorganisms to use glutamate as a nitrogen source effectiveness. Therefore, in addition to the exogenous nucleic acid sequence encoding the naphthalene dioxygenase, the genetically modified microorganism of the present invention can also be further deleted from its endogenous icd gene to further promote the utilization efficiency of glutamine by the microorganism. .
在一特定實施例中，上述本發明之經基因改質的微生物可為民國107年11月22日寄存於中華民國食品工業發展研究所生物資源保存及研究中心，寄存編號為BCRC 940687的大腸桿菌基因改植株JD938。In a specific embodiment, the above-mentioned genetically modified microorganism of the present invention can be deposited with the Bioresource Conservation and Research Center of the Food Industry Development Research Institute of the Republic of China on November 22, 2007, and the deposit number is BCRC 940687. Genetically modified plant JD938.
本發明也提供一種新穎之大腸桿菌基因改質株，其為於民國107年11月22日寄存於中華民國食品工業發展研究所生物資源保存及研究中心，寄存編號為BCRC 940687的大腸桿菌基因改質株JD938。The present invention also provides a novel genetically modified strain of Escherichia coli, which was deposited at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China on November 22, 1987, and the deposit number is BCRC 940687. Plasmid strain JD938.
本發明也提供一種製備經基因改質的微生物的方法，其可包括以下步驟，但不限於此。The present invention also provides a method for preparing a genetically modified microorganism, which may include the following steps, but is not limited thereto.
首先，將一微生物之內源性 icd基因剔除以獲得一經 icd基因剔除的微生物。 Firstly, the endogenous icd gene of a microorganism is deleted to obtain an icd gene deleted microorganism.
將上述微生物之內源性 icd基因剔除的方法，並無特殊限制，只要可使標的內源性 icd基因失活，且不影響非標的基因的表現即可。例如，可以該技術領域中具有通常知識者所熟知之任何剔除內源性標的基因的方法進行，如同源序列互換(homologous recombination)、噬菌體導入(phage transduction)、常間回文重複序列叢集-半胱氨酸蛋白酶9系統(Clustered Regularly Interspaced Short Palindromic Repeats- Cysteine asparate protease (Caspase) 9, CRISPER-Cas9 system)等，但不限於此。 There are no special restrictions on the method for removing the endogenous icd genes of the above-mentioned microorganisms, as long as the target endogenous icd genes can be inactivated without affecting the performance of non-target genes. For example, it can be performed by any method known to those with ordinary knowledge in the technical field to remove the endogenous target gene, such as homologous recombination, phage transduction, and frequent palindromic repetitive sequence cluster-half. The cystine protease 9 system (Clustered Regularly Interspaced Short Palindromic Repeats-Cysteine asparate protease (Caspase) 9, CRISPER-Cas9 system), but not limited to this.
上述微生物可包括，但不限於細菌或真菌。The aforementioned microorganisms may include, but are not limited to bacteria or fungi.
關於上述細菌的例子與相關說明，可為與上方本發明之經基因改質的微生物之相關段落中所記載之細菌的例子與相關說明相同，故不於此重複以避免贅述。The examples and related descriptions of the above-mentioned bacteria can be the same as those described in the relevant paragraphs of the genetically modified microorganisms of the present invention above, so they will not be repeated here to avoid repetition.
相似地，關於上述真菌的例子與相關說明，也可為與上方本發明之經基因改質的微生物之相關段落中所記載之真菌的例子與相關說明相同，故不於此重複以避免贅述。Similarly, the examples and related descriptions of the above-mentioned fungi can also be the same as the examples and related descriptions of the fungi described in the relevant paragraphs of the genetically modified microorganisms of the present invention above, so they will not be repeated here to avoid repetition.
接著，以含有麩胺酸及/或其鹽類的培養基對上述經 icd基因剔除的微生物進行馴化，以獲得一經馴化之微生物，而上述經馴化之微生物的麩胺酸利用率可高於上述經 icd基因剔除的微生物。於此培養基中，麩胺酸及/或其鹽類之濃度可為約0.05-80 g/L，例如，約0.1-80 g/L、約0.1-70 g/L、約0.1-60 g/L、約0.5-55 g/L、約0.5-40 g/L、約1-50 g/L，約1-30 g/L、約5-20 g/L、約10-30 g/L、約15-40 g/L、約5-45 g/L等，但不限於此。 Next, the above-mentioned icd gene knocked- out microorganisms are domesticated with a medium containing glutamate and/or its salts to obtain a domesticated microorganism, and the utilization rate of glutamate of the domesticated microorganism can be higher than that of the above-mentioned icd gene knocked out microorganisms. In this medium, the concentration of glutamic acid and/or its salts can be about 0.05-80 g/L, for example, about 0.1-80 g/L, about 0.1-70 g/L, about 0.1-60 g/L L, about 0.5-55 g/L, about 0.5-40 g/L, about 1-50 g/L, about 1-30 g/L, about 5-20 g/L, about 10-30 g/L, About 15-40 g/L, about 5-45 g/L, etc., but not limited thereto.
在一實施例中，上述含有麩胺酸及/或其鹽類的培養基可更包含M9鹽類溶液。In one embodiment, the above-mentioned medium containing glutamate and/or its salts may further include M9 salt solution.
M9鹽類溶液包括Na 2HPO 4·2H 2O、KH 2PO 4、NaCl與NH 4Cl。上述M9鹽類溶液可為1倍(1X)之M9鹽類溶液，但不限於此。於1倍(1X)之M9鹽類溶液中，Na 2HPO 4·2H 2O之濃度為約7.52 g/L、KH 2PO 4之濃度為約3.0 g/L、NaCl之濃度為約0.5 g/L、而NH 4Cl之濃度為約0.5 g/L。 The M9 salt solution includes Na 2 HPO 4 ·2H 2 O, KH 2 PO 4 , NaCl and NH 4 Cl. The above-mentioned M9 salt solution can be a 1X (1X) M9 salt solution, but it is not limited thereto. In a 1X (1X) M9 salt solution, the concentration of Na 2 HPO 4 ·2H 2 O is about 7.52 g/L, the concentration of KH 2 PO 4 is about 3.0 g/L, and the concentration of NaCl is about 0.5 g /L, and the concentration of NH 4 Cl is about 0.5 g/L.
又在一實施例中，上述含有麩胺酸及/或其鹽類的培養基可更包含一碳源。碳源的種類並無特殊限制，只要是可被所培養之微生物作為碳源利用即可，例如，甘油、葡萄糖、乳糖等，但不限於此。於上述含有麩胺酸及/或其鹽類的培養基中，碳源的濃度可為約0.05-100 g/L，例如，約0.05-15 g/L、0.1-20 g/L、0.5-15 g/L、1-10 g/L、5-20 g/L、5-30 g/L、10-50 g/L、20-60 g/L、20-80 g/L、30-100 g/L等，但不限於此。在一特定實施例中，上述碳源為甘油，而於上述含有麩胺酸及/或其鹽類的培養基中，甘油的濃度可為約0.05-80 g/L，例如，約0.05-15 g/L、0.1-20 g/L、0.5-15 g/L、1-10 g/L、1-20 g/L、1-30 g/L、5-40 g/L、5-50 g/L、10-60 g/L、15-70 g/L、20-80 g/L等。In another embodiment, the aforementioned medium containing glutamic acid and/or its salts may further include a carbon source. The type of carbon source is not particularly limited, as long as it can be used as a carbon source by the cultured microorganisms, for example, glycerol, glucose, lactose, etc., but not limited thereto. In the above-mentioned medium containing glutamic acid and/or its salts, the concentration of the carbon source may be about 0.05-100 g/L, for example, about 0.05-15 g/L, 0.1-20 g/L, 0.5-15 g/L, 1-10 g/L, 5-20 g/L, 5-30 g/L, 10-50 g/L, 20-60 g/L, 20-80 g/L, 30-100 g /L etc., but not limited to this. In a specific embodiment, the carbon source is glycerol, and in the medium containing glutamate and/or its salts, the concentration of glycerol may be about 0.05-80 g/L, for example, about 0.05-15 g /L, 0.1-20 g/L, 0.5-15 g/L, 1-10 g/L, 1-20 g/L, 1-30 g/L, 5-40 g/L, 5-50 g/ L, 10-60 g/L, 15-70 g/L, 20-80 g/L, etc.
在一實施例中，上述以含有麩胺酸及/或其鹽類的培養基對上述經 icd基因剔除的微生物進行的馴化，可包括，但不限於，進行一第一濃度培養。而於第一濃度培養中，將上述經 icd基因剔除的微生物以含有一第一濃度之麩胺酸及/或其鹽類的培養基培養至生長穩定期。上述麩胺酸及/或其鹽類於培養基中之第一濃度可為約0.05-80 g/L，例如，約0.1-80 g/L、約0.1-70 g/L、約0.1-60 g/L、約0.5-55 g/L、約0.5-40 g/L、約1-50 g/L，約1-30 g/L、約5-20 g/L、約10-30 g/L、約15-40 g/L、約5-45 g/L等，但不限於此。在一實施例中，於上述含有麩胺酸及/或其鹽類之培養基中，麩胺酸及/或其鹽類的濃度可為約1 g/L、5 g/L、10 g/L、15 g/L、20 g/L、30 g/L、40 g/L、50 g/L、60 g/L、70 g/L或80 g/L，但不限於此。又，上述第一濃度培養視需要而定，可僅進行一次，也可進行複數次之循環，並無特殊限制。而在一特定實施例中，上述第一濃度培養可進行約3-6個循環，但不限於此，操作者可視實際情況調整循環次數。 In one embodiment, the above-mentioned domestication of the above-mentioned icd gene knocked- out microorganism with a medium containing glutamic acid and/or its salt may include, but is not limited to, culturing at a first concentration. In the first concentration culture, the above-mentioned icd gene knocked- out microorganisms are cultured in a medium containing a first concentration of glutamic acid and/or its salts to a stable growth period. The first concentration of the above-mentioned glutamic acid and/or its salt in the medium can be about 0.05-80 g/L, for example, about 0.1-80 g/L, about 0.1-70 g/L, about 0.1-60 g /L, about 0.5-55 g/L, about 0.5-40 g/L, about 1-50 g/L, about 1-30 g/L, about 5-20 g/L, about 10-30 g/L , About 15-40 g/L, about 5-45 g/L, etc., but not limited to this. In one embodiment, in the medium containing glutamate and/or its salts, the concentration of glutamate and/or its salts may be about 1 g/L, 5 g/L, 10 g/L , 15 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L or 80 g/L, but not limited to this. In addition, the above-mentioned first concentration culture depends on need, and it may be performed only once, or may be performed multiple times, and there is no particular limitation. In a specific embodiment, the above-mentioned first concentration culture can be carried out for about 3-6 cycles, but it is not limited to this, and the operator can adjust the number of cycles according to the actual situation.
此外，在上述第一濃度培養中，將微生物培養至生長穩定期的時間係根據微生物之種類、培養條件等而定，也無特殊限制。例如，在上述第一濃度培養中可將微生物培養約12-84小時，例如，約12-24小時，約36-48小時，約48-60小時，約60-72小時，約24小時、約48小時，約72小時等，以至生長穩定期，但不限於此。In addition, in the above-mentioned first concentration culture, the time for cultivating the microorganisms to the stable growth period depends on the type of microorganisms, culture conditions, etc., and there is no particular limitation. For example, the microorganisms can be cultured for about 12-84 hours in the above-mentioned first concentration culture, for example, about 12-24 hours, about 36-48 hours, about 48-60 hours, about 60-72 hours, about 24 hours, about 48 hours, about 72 hours, etc., until the stable growth period, but not limited to this.
再者，於第一濃度培養中，培養微生物所採用之溫度，也同樣根據微生物之種類、培養條件等而定，也無特殊限制。例如，在上述第一濃度培養中，可將微生物培養於約25-40 oC，例如，約25-30 oC、約30-35 oC、約30-40 oC、約28 oC、約30 oC、約32 oC、約37 oC、約40 oC，但不限於此。 Furthermore, in the first concentration culture, the temperature used for culturing the microorganisms is also determined according to the type of microorganisms, culture conditions, etc., and there is no special restriction. For example, in the first concentration in the culture, the microorganisms may be cultured at about 25-40 o C, e.g., about 25-30 o C, about 30-35 o C, about 30-40 o C, from about 28 o C, About 30 o C, about 32 o C, about 37 o C, about 40 o C, but not limited to this.
而在另一實施例中，上述以含有麩胺酸及/或其鹽類的培養基對上述經 icd基因剔除的微生物進行的馴化，除了上述第一濃度培養外，可更包括，但不限於，於上述第一濃度培養之後，對上述 icd基因剔除的微生物進行至少一回合之增加濃度培養，而於每回合之增加濃度培養中，將上述經 icd基因剔除的微生物以含有高於上述第一濃度之濃度的麩胺酸及/或其鹽類的培養基培養至生長穩定期。又，每回合之增加濃度培養所使用之培養基的麩胺酸及/或其鹽類濃度皆為高於前一回合之增加濃度培養。 In another embodiment, the above-mentioned domestication of the above-mentioned icd gene knocked- out microorganism with a medium containing glutamic acid and/or its salt may further include, but is not limited to, in addition to the above-mentioned first concentration culture, After the above-mentioned first concentration of culture, the above-mentioned icd gene knocked out microorganisms are cultured at an increased concentration for at least one round, and in each round of increased concentration culture, the above-mentioned icd gene knocked out microorganisms are cultured at a higher concentration than the above-mentioned first concentration The culture medium of glutamic acid and/or its salt is cultivated to the stable growth period. In addition, the concentration of glutamic acid and/or its salt in the medium used in each round of increasing concentration culture is higher than that of the previous round of increasing concentration culture.
又，上述至少一回合之增加濃度培養的數目並無特殊限制，可視微生物之生理情況，例如麩胺酸之利用效率而定。舉例來說，倘若微生物對麩胺酸之利用效率低或不如預期，則可接續進行增加濃度培養。相反地，倘若微生物對麩胺酸之利用效率高或已達預期，此時則可停止後續之增加濃度培養。在一實施例中，於上述第一濃度培養之後，可進行約1-5回合之增加濃度培養，例如可接續進行3回合之增加濃度培養，但不限於此。In addition, there is no special limit to the number of the above-mentioned at least one round of increased concentration culture, which may be determined by the physiological condition of the microorganism, such as the utilization efficiency of glutamic acid. For example, if the utilization efficiency of glutamic acid by microorganisms is low or not as expected, the culture can be continued to increase the concentration. Conversely, if the utilization efficiency of glutamic acid by the microorganisms is high or has reached the expectation, then the subsequent increasing concentration culture can be stopped. In one embodiment, after the above-mentioned first concentration cultivation, about 1-5 rounds of increasing concentration cultivation may be performed, for example, 3 rounds of increasing concentration cultivation may be continued, but it is not limited to this.
此外，上述每回合之增加濃度培養為獨立地視需要而定，每回合之增加濃度培養可獨立地僅進行一次，也可進行複數次之循環，並無特殊限制。而在一特定實施例中，上述每回合之增加濃度培養可獨立地進行約3-6個循環，但不限於此，操作者可視實際情況調整循環次數。In addition, the above-mentioned increasing concentration culture for each round is independently determined as needed, and each round of increasing concentration cultivation may be independently performed only once, or may be repeated multiple times, without special restrictions. In a specific embodiment, each round of the above-mentioned increasing concentration culture can be carried out independently for about 3-6 cycles, but it is not limited to this, and the operator can adjust the number of cycles according to the actual situation.
又，於上述製備用於生產靛藍染料之經基因改質的微生物的方法中，在以含有麩胺酸及/或其鹽類的培養基對上述經 icd基因剔除的微生物進行馴化，以獲得一經馴化之微生物之後，將編碼萘二氧酶之一外源性核酸序列導入上述經馴化之微生物，以獲得用於生產靛藍染料之經基因改質的微生物。 In addition, in the above-mentioned method of preparing genetically modified microorganisms for producing indigo dye, the above-mentioned icd gene knocked- out microorganisms are domesticated with a medium containing glutamic acid and/or its salts to obtain a domesticated After the microorganism, an exogenous nucleic acid sequence encoding naphthalene dioxygenase is introduced into the above-mentioned domesticated microorganism to obtain a genetically modified microorganism for the production of indigo dye.
將上述外源性核酸序列導入上述經馴化之微生物之方式並無特別限制，只要可將上述外源性核酸序列導入上述經馴化之微生物並可使其表現即可。例如，可經由一載體將上述外源性核酸序列導入上述經馴化之微生物中，但不限於此。又，上述載體之種類可依據所實際採用之微生物所適合的載體種類而定，例如可以特定質體(plasmid)作為載體，但不限於此。另外，也可透過將外源性核酸序列送入菌株之基因體中使其表現。在一實施例中，可經由一質體將上述外源性核酸序列導入上述經馴化之微生物中。The method of introducing the above-mentioned exogenous nucleic acid sequence into the above-mentioned domesticated microorganism is not particularly limited, as long as the above-mentioned exogenous nucleic acid sequence can be introduced into the above-mentioned domesticated microorganism and can be expressed. For example, the above-mentioned exogenous nucleic acid sequence can be introduced into the above-mentioned domesticated microorganism via a vector, but it is not limited thereto. In addition, the type of the above-mentioned carrier can be determined according to the type of carrier suitable for the microorganism actually used, for example, a specific plasmid can be used as the carrier, but it is not limited thereto. In addition, it can also be expressed by sending exogenous nucleic acid sequences into the genome of the strain. In one embodiment, the aforementioned exogenous nucleic acid sequence can be introduced into the aforementioned domesticated microorganism via a plastid.
上述外源性核酸序列可包括與序列辨識號：1之序列具有至少85%之序列相似度的一序列，但不限於此。在一實施例中，上述外源性核酸序列可包括序列辨識號：1之序列。在另一實施例中，上述外源性核酸序列可為序列辨識號：1之序列。The aforementioned exogenous nucleic acid sequence may include a sequence with at least 85% sequence similarity to the sequence of sequence identification number: 1, but is not limited thereto. In one embodiment, the above-mentioned exogenous nucleic acid sequence may include a sequence of sequence identification number: 1. In another embodiment, the above-mentioned exogenous nucleic acid sequence may be a sequence with sequence identification number: 1.
在一特定實施例中，於上述本發明之製備經基因改質的微生物的方法中，所述經基因改質的微生物可為一經基因改質的大腸桿菌，其於民國107年11月22日寄存於中華民國食品工業發展研究所生物資源保存及研究中心，寄存編號為BCRC 940687。In a specific embodiment, in the method for preparing a genetically modified microorganism of the present invention, the genetically modified microorganism may be a genetically modified Escherichia coli, which was published on November 22, 2007 Deposited at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China, and the deposit number is BCRC 940687.
另外，依據上述，本發明還可提供藉由任何上述之本發明之製備經基因改質的微生物的方法所製備出之任何經基因改質的微生物。In addition, according to the above, the present invention can also provide any genetically modified microorganisms prepared by any of the above-mentioned methods of the present invention for preparing genetically modified microorganisms.
在一實施例中，上述之本發明之任何製備經基因改質的微生物的方法所製備出之任何經基因改質的微生物，係可用於生產靛藍染料，但不限於此。In one embodiment, any genetically modified microorganism prepared by any method for preparing genetically modified microorganism of the present invention described above can be used to produce indigo dye, but is not limited thereto.
再者，本發明也可提供一種生產靛藍染料的方法。Furthermore, the present invention can also provide a method for producing indigo dye.
本發明之生產靛藍染料的方法可包括以下步驟，但不限於此。The method for producing indigo dye of the present invention may include the following steps, but is not limited thereto.
於本發明生產靛藍染料的方法中，可將含有編碼萘二氧酶之一外源性核酸序列的一經基因改質之微生物，培養於含有麩胺酸及/或其鹽類與靛藍染料前驅物之一培養基中，以使上述經基因改質的微生物以上述麩胺酸及/或其鹽類為氮源以進行生長，並以靛藍染料前驅物為基質以產生靛藍染料。In the method for producing indigo dye of the present invention, a genetically modified microorganism containing an exogenous nucleic acid sequence encoding naphthalene dioxygenase can be cultured on a precursor containing glutamic acid and/or its salt and indigo dye precursor In a medium, the genetically modified microorganisms are grown using the glutamic acid and/or salts thereof as a nitrogen source, and the indigo dye precursor is used as a substrate to produce indigo dye.
而上述含有編碼萘二氧酶之一外源性核酸序列的一經基因改質之微生物的例子，可包括，但不限於，上述任何之本發明之經基因改質的微生物、上述本發明之新穎之大腸桿菌基因改質株、或藉由任何上述之本發明之製備經基因改質的微生物的方法所製備出之任何經基因改質的微生物。The above examples of a genetically modified microorganism containing an exogenous nucleic acid sequence encoding a naphthalene dioxygenase may include, but are not limited to, any of the above-mentioned genetically modified microorganisms of the present invention, and the above-mentioned novelty of the present invention. E. coli genetically modified strain, or any genetically modified microorganism prepared by any of the above-mentioned methods of the present invention for preparing genetically modified microorganisms.
又，於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中，麩胺酸及/或其鹽類的濃度可為約0.05-80 g/L，例如，約0.1-80 g/L、約0.1-70 g/L、約0.1-60 g/L、約0.5-55 g/L、約0.5-40 g/L、約1-50 g/L，約1-30 g/L、約5-20 g/L、約10-30 g/L、約15-40 g/L、約5-45 g/L等，但不限於此。在一實施例中，於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中，麩胺酸及/或其鹽類的濃度可為約1 g/L、5 g/L、10 g/L、15 g/L、20 g/L、30 g/L、40 g/L、50 g/L、60 g/L、70 g/L或80 g/L。In addition, in the aforementioned medium containing glutamic acid and/or its salts and indigo dye precursors, the concentration of glutamic acid and/or its salts may be about 0.05-80 g/L, for example, about 0.1-80 g/L, about 0.1-70 g/L, about 0.1-60 g/L, about 0.5-55 g/L, about 0.5-40 g/L, about 1-50 g/L, about 1-30 g/ L, about 5-20 g/L, about 10-30 g/L, about 15-40 g/L, about 5-45 g/L, etc., but not limited thereto. In one embodiment, in the medium containing glutamic acid and/or its salt and indigo dye precursor, the concentration of glutamic acid and/or its salt may be about 1 g/L, 5 g/L , 10 g/L, 15 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L or 80 g/L.
上述靛藍染料前驅物的例子，可包括色胺酸、吲哚等，但不限於此。又，於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中，上述靛藍染料前驅物的濃度可為約0.1-25 g/L，例如約0.5-20 g/L、約1-15 g/L、約2-12 g/L等。在一實施例中，上述靛藍染料前驅物的濃度可為約3-10 g/L。在一實施例中，上述靛藍染料前驅物可為色胺酸，而於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中，色胺酸的濃度可為約0.1-25 g/L，例如，約0.1-20 g/L、約1-20 g/L、約2-18 g/L、約3-15 g/L、約3-10 g/L、約5-15 g/L等，但不限於此。Examples of the aforementioned indigo dye precursors may include tryptophan, indole, etc., but are not limited thereto. In addition, in the medium containing glutamic acid and/or its salt and indigo dye precursor, the concentration of the indigo dye precursor may be about 0.1-25 g/L, such as about 0.5-20 g/L, about 1-15 g/L, about 2-12 g/L, etc. In one embodiment, the concentration of the aforementioned indigo dye precursor may be about 3-10 g/L. In one embodiment, the indigo dye precursor may be tryptophan, and in the medium containing glutamine and/or its salt and indigo dye precursor, the concentration of tryptophan may be about 0.1-25 g/L, for example, about 0.1-20 g/L, about 1-20 g/L, about 2-18 g/L, about 3-15 g/L, about 3-10 g/L, about 5-15 g/L etc., but not limited to this.
在一實施例中，上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基，可更包含一酵母菌萃取物。酵母菌萃取物於上述含有麩胺酸及/或其鹽類的培養基中的濃度可為約0.05-30 g/L，例如，約0.1-30 g/L、約0.5-25 g/L、約1-20 g/L，約2-18 g/L、約3-15 g/L、約3-10 g/L、約5-20 g/L等，但不限於此。在一實施例中，酵母菌萃取物於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中的濃度可為約1 g/L、3 g/L、5 g/L 、20 g/L或30 g/L。In one embodiment, the above-mentioned medium containing glutamic acid and/or its salt and indigo dye precursor may further include a yeast extract. The concentration of the yeast extract in the medium containing glutamine and/or its salts can be about 0.05-30 g/L, for example, about 0.1-30 g/L, about 0.5-25 g/L, about 1-20 g/L, about 2-18 g/L, about 3-15 g/L, about 3-10 g/L, about 5-20 g/L, etc., but not limited thereto. In one embodiment, the concentration of the yeast extract in the medium containing glutamic acid and/or its salt and indigo dye precursor may be about 1 g/L, 3 g/L, 5 g/L, 20 g/L or 30 g/L.
在另一實施例中，上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基可更包含M9鹽類溶液、硫胺(thiamine)、甘油、CaCl 2、MgSO 4與異丙基-β-D-硫代半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside, IPTG)。 In another embodiment, the above-mentioned medium containing glutamic acid and/or its salt and indigo dye precursor may further include M9 salt solution, thiamine, glycerol, CaCl 2 , MgSO 4 and isopropyl -β-D-thiogalactopyranoside (Isopropyl β-D-1-thiogalactopyranoside, IPTG).
M9鹽類溶液包括Na 2HPO 4·2H 2O、KH 2PO 4、NaCl與NH 4Cl。上述M9鹽類溶液可為1倍(1X)之M9鹽類溶液，但不限於此。於1倍(1X)之M9鹽類溶液中，Na 2HPO 4·2H 2O之濃度為約7.52 g/L，KH 2PO 4之濃度為約3.0 g/L、NaCl之濃度為約0.5 g/L，而NH 4Cl之濃度為約0.5 g/L。 The M9 salt solution includes Na 2 HPO 4 ·2H 2 O, KH 2 PO 4 , NaCl and NH 4 Cl. The above-mentioned M9 salt solution can be a 1X (1X) M9 salt solution, but it is not limited thereto. In a 1X (1X) M9 salt solution, the concentration of Na 2 HPO 4 ·2H 2 O is about 7.52 g/L, the concentration of KH 2 PO 4 is about 3.0 g/L, and the concentration of NaCl is about 0.5 g /L, and the concentration of NH 4 Cl is about 0.5 g/L.
又，硫胺於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中的濃度可為約0.05-20 mg/L，例如，約0.05-10 mg/L、約0.1-15 mg/L、約0.1-1 mg/L、約0.5-8 mg/L、約1-5 mg/L等，但不限於此。甘油於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中的濃度可為約0.05-80 g/L，例如，約0.05-15 g/L、0.1-20 g/L、0.5-15 g/L、1-10 g/L、1-20 g/L、1-30 g/L、5-40 g/L、5-50 g/L、10-60 g/L、15-70 g/L、20-80 g/L等，但不限於此。MgSO 4於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中的濃度可為約0.05-20 mM，例如，約0.05-10 mM、約0.1-15 mM、約0.1-1 mM、約0.5-8 mM、約1-5 mM等，但不限於此。又，CaCl 2於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中的濃度可為約0.05-20 μM，例如，約0.05-10 μM、約0.1-15 μM、約0.1-10 μM、約0.5-8 μM、約1-5 μM等，但不限於此。而異丙基-β-D-硫代半乳糖苷於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中的濃度可為約0.005-10 mM，例如，約0.005-5 mM、約0.01-10 mM、約0.01-1 mM、約0.1-10 mM、約0.1-5 mM、約0.5-5 mM等，但也不限於此。 In addition, the concentration of thiamine in the medium containing glutamic acid and/or its salt and indigo dye precursor may be about 0.05-20 mg/L, for example, about 0.05-10 mg/L, about 0.1-15 mg/L, about 0.1-1 mg/L, about 0.5-8 mg/L, about 1-5 mg/L, etc., but not limited thereto. The concentration of glycerol in the medium containing glutamine and/or its salt and indigo dye precursor can be about 0.05-80 g/L, for example, about 0.05-15 g/L, 0.1-20 g/L, 0.5-15 g/L, 1-10 g/L, 1-20 g/L, 1-30 g/L, 5-40 g/L, 5-50 g/L, 10-60 g/L, 15 -70 g/L, 20-80 g/L, etc., but not limited to this. The concentration of MgSO 4 in the above-mentioned medium containing glutamic acid and/or its salt and indigo dye precursor may be about 0.05-20 mM, for example, about 0.05-10 mM, about 0.1-15 mM, about 0.1-1 mM, about 0.5-8 mM, about 1-5 mM, etc., but not limited thereto. In addition, the concentration of CaCl 2 in the above-mentioned medium containing glutamic acid and/or its salt and indigo dye precursor can be about 0.05-20 μM, for example, about 0.05-10 μM, about 0.1-15 μM, about 0.1 -10 μM, about 0.5-8 μM, about 1-5 μM, etc., but not limited thereto. The concentration of isopropyl-β-D-thiogalactoside in the above-mentioned medium containing glutamic acid and/or its salt and indigo dye precursor may be about 0.005-10 mM, for example, about 0.005-5 mM, about 0.01-10 mM, about 0.01-1 mM, about 0.1-10 mM, about 0.1-5 mM, about 0.5-5 mM, etc., but not limited thereto.
在又另一實施例中，上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基除了可更包含M9鹽類溶液、硫胺(thiamine)、甘油、CaCl 2、MgSO 4與異丙基-β-D-硫代半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside, IPTG)以外，還可進一步包含一酵母菌萃取物。酵母菌萃取物於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中的濃度可為約0.05-30 g/L，例如，約0.1-30 g/L、約0.5-25 g/L、約1-20 g/L、約2-18 g/L、約3-15 g/L、約3-10 g/L、約5-20 g/L等，但不限於此。在一實施例中，酵母菌萃取物於上述含有麩胺酸及/或其鹽類與靛藍染料前驅物之培養基中的濃度可為約1 g/L、3 g/L、5 g/L或20 g/L。 In yet another embodiment, the above-mentioned medium containing glutamate and/or its salts and indigo dye precursors may further include M9 salt solution, thiamine, glycerin, CaCl 2 , MgSO 4 and isoforms. In addition to propyl-β-D-thiogalactopyranoside (IPTG), it may further include a yeast extract. The concentration of the yeast extract in the above-mentioned medium containing glutamic acid and/or its salt and indigo dye precursor may be about 0.05-30 g/L, for example, about 0.1-30 g/L, about 0.5-25 g/L, about 1-20 g/L, about 2-18 g/L, about 3-15 g/L, about 3-10 g/L, about 5-20 g/L, etc., but not limited thereto. In one embodiment, the concentration of the yeast extract in the medium containing glutamic acid and/or its salt and indigo dye precursor may be about 1 g/L, 3 g/L, 5 g/L or 20 g/L.
此外，於本發明生產靛藍染料之方法中，培養上述經基因改質之微生物的時間係根據微生物之種類、培養情況等而定，並無特殊限制。例如，可將上述經基因改質之微生物培養約12-84小時，例如，約12-24小時，約36-48小時，約48-60小時，約60-72小時，約24小時、約48小時，約72小時等，以至生長穩定期，但不限於此。 In addition, in the method for producing indigo dye of the present invention, the time for cultivating the above-mentioned genetically modified microorganisms depends on the type of microorganisms, culture conditions, etc., and is not particularly limited. For example, the above-mentioned genetically modified microorganism can be cultured for about 12-84 hours, for example, about 12-24 hours, about 36-48 hours, about 48-60 hours, about 60-72 hours, about 24 hours, about 48 hours. Hours, about 72 hours, etc., until the stable growth period, but not limited to this.
再者，於本發明生產靛藍染料之方法中，培養上述經基因改質之微生物所採用之溫度，也同樣根據微生物之種類、培養情況等而定，也無特殊限制。例如，於本發明生產靛藍染料之方法中，可將微生物培養於約25-40℃，例如，約25-30℃、約30-35℃、約30-40℃、約28℃、約30℃、約32℃、約37℃、約40℃，但不限於此。 Furthermore, in the method for producing indigo dye of the present invention, the temperature used for culturing the above-mentioned genetically modified microorganisms also depends on the type of microorganisms, culture conditions, etc., and there is no particular limitation. For example, in the method for producing indigo dye of the present invention, microorganisms can be cultured at about 25-40°C, for example, about 25-30°C, about 30-35°C, about 30-40°C, about 28°C, or about 30°C. , About 32°C, about 37°C, about 40°C, but not limited thereto.
藉由上述任何之本發明生產靛藍染料之方法，不僅可使微生物有效利用麩胺酸，並可有效提升菌體之靛藍染料生產效率以及菌體中靛藍染料之含量。 By any of the above-mentioned methods for producing indigo dye of the present invention, not only can microorganisms effectively utilize glutamic acid, but also can effectively increase the production efficiency of indigo dye in the bacterial cell and the indigo dye content in the bacterial cell.
本發明還提供一種培養基用於培養一經基因改質的微生物並使其生產靛藍染料的用途。 The invention also provides the use of a culture medium for culturing a genetically modified microorganism and making it produce indigo dye.
上述培養基可包括麩胺酸及/或其鹽類，但不限於此。於上述培養基中，麩胺酸及/或其鹽類的濃度可為約0.05-80g/L，例如，約0.1-60g/L、約0.5-55g/L、約0.5-40g/L、約1-50g/L、約1-30g/L、約5-20g/L、約10-30g/L、約15-40g/L、約5-45g/L等，但不限於此。在一實施例中，於上述培養基中，麩胺酸及/或其鹽類的濃度可為約1g/L、5g/L、10g/L、15g/L、20g/L、30g/L、40g/L或50g/L。 The aforementioned culture medium may include glutamic acid and/or its salts, but is not limited thereto. In the above medium, the concentration of glutamine and/or its salts may be about 0.05-80 g/L, for example, about 0.1-60 g/L, about 0.5-55 g/L, about 0.5-40 g/L, about 1 -50g/L, about 1-30g/L, about 5-20g/L, about 10-30g/L, about 15-40g/L, about 5-45g/L, etc., but not limited thereto. In one embodiment, the concentration of glutamic acid and/or its salts in the above medium may be about 1g/L, 5g/L, 10g/L, 15g/L, 20g/L, 30g/L, 40g /L or 50g/L.
上述培養基除了麩胺酸及/或其鹽類之外，還可更包括一靛藍染料前驅物。上述靛藍染料前驅物的例子，可包括色胺酸、吲哚等，但不限於此。又，於上述培養基中，上述靛藍染料前驅物的濃度可為約0.1-25 g/L，例如約0.5-20 g/L、約1-15 g/L、約2-12 g/L等。在一實施例中，上述靛藍染料前驅物的濃度可為約3-10 g/L。在一實施例中，上述靛藍染料前驅物為色胺酸，而於上述培養基中，色胺酸的濃度可為約0.1-25 g/L，例如，約0.1-20 g/L、約1-20 g/L、約2-18 g/L、約3-15 g/L、約3-10 g/L、約5-15 g/L等，但不限於此。In addition to glutamic acid and/or its salts, the above-mentioned culture medium may further include an indigo dye precursor. Examples of the aforementioned indigo dye precursors may include tryptophan, indole, etc., but are not limited thereto. In addition, in the medium, the concentration of the indigo dye precursor may be about 0.1-25 g/L, for example, about 0.5-20 g/L, about 1-15 g/L, about 2-12 g/L, etc. In one embodiment, the concentration of the aforementioned indigo dye precursor may be about 3-10 g/L. In one embodiment, the precursor of the indigo dye is tryptophan, and in the medium, the concentration of tryptophan may be about 0.1-25 g/L, for example, about 0.1-20 g/L, about 1- 20 g/L, about 2-18 g/L, about 3-15 g/L, about 3-10 g/L, about 5-15 g/L, etc., but not limited thereto.
上述培養基可更包含一碳源。碳源的種類並無特殊限制，只要是可被所培養之微生物作為碳源利用即可，例如，甘油、葡萄糖、乳糖等，但不限於此。於上述培養基中，碳源的濃度可為約0.05-100 g/L，例如，約0.05-15 g/L、0.1-20 g/L、0.5-15 g/L、1-10 g/L、5-20 g/L、5-30 g/L、10-50 g/L、20-60 g/L、20-80 g/L、30-100 g/L等，但不限於此。在一特定實施例中，上述碳源為甘油，而於上述培養基中，甘油的濃度可為約0.05-80 g/L，例如，約0.05-15 g/L、0.1-20 g/L、0.5-15 g/L、1-10 g/L、1-20 g/L、1-30 g/L、5-40 g/L、5-50 g/L、10-60 g/L、15-70 g/L、20-80 g/L等，但不限於此。The above-mentioned medium may further contain a carbon source. The type of carbon source is not particularly limited, as long as it can be used as a carbon source by the cultured microorganisms, for example, glycerol, glucose, lactose, etc., but not limited thereto. In the above medium, the concentration of the carbon source can be about 0.05-100 g/L, for example, about 0.05-15 g/L, 0.1-20 g/L, 0.5-15 g/L, 1-10 g/L, 5-20 g/L, 5-30 g/L, 10-50 g/L, 20-60 g/L, 20-80 g/L, 30-100 g/L, etc., but not limited to this. In a specific embodiment, the carbon source is glycerol, and in the medium, the concentration of glycerol may be about 0.05-80 g/L, for example, about 0.05-15 g/L, 0.1-20 g/L, 0.5 -15 g/L, 1-10 g/L, 1-20 g/L, 1-30 g/L, 5-40 g/L, 5-50 g/L, 10-60 g/L, 15- 70 g/L, 20-80 g/L, etc., but not limited to this.
又，在一實施例中，上述培養基除了麩胺酸及/或其鹽類之外，還可更包括一酵母菌萃取物。酵母菌萃取物於上述培養基中的濃度可為約0.05-30 g/L，例如，約0.1-30 g/L、約0.5-25 g/L、約1-20 g/L、約2-18 g/L、約3-15 g/L、約3-10 g/L、約5-20 g/L等，但不限於此。在一特定實施例中，酵母菌萃取物於上述培養基中的濃度可為約1 g/L、3 g/L、5 g/L或20 g/L。Furthermore, in one embodiment, the above-mentioned culture medium may further include a yeast extract in addition to glutamate and/or its salts. The concentration of the yeast extract in the above medium may be about 0.05-30 g/L, for example, about 0.1-30 g/L, about 0.5-25 g/L, about 1-20 g/L, about 2-18 g/L, about 3-15 g/L, about 3-10 g/L, about 5-20 g/L, etc., but not limited thereto. In a specific embodiment, the concentration of the yeast extract in the aforementioned medium may be about 1 g/L, 3 g/L, 5 g/L or 20 g/L.
在另一實施例中，上述培養基除了麩胺酸及/或其鹽類之外，還可更包括M9鹽類溶液、硫胺、甘油、CaCl 2、MgSO 4與異丙基-β-D-硫代半乳糖苷。 In another embodiment, in addition to glutamine and/or its salts, the above-mentioned culture medium may further include M9 salt solution, thiamine, glycerol, CaCl 2 , MgSO 4 and isopropyl-β-D- Thiogalactoside.
關於M9鹽類溶液與其各成分之說明，可為與上方本發明生產靛藍染料之方法之相關段落中所記載之M9鹽類溶液之相關說明相同，故不於此重複以避免贅述。The description of the M9 salt solution and its components can be the same as the relevant description of the M9 salt solution described in the relevant paragraph of the method for producing indigo dye of the present invention, so it will not be repeated here to avoid repetition.
相似地，關於硫胺、甘油、CaCl 2、MgSO 4與異丙基-β-D-硫代半乳糖苷之濃度說明，也可為與上方本發明生產靛藍染料之方法之相關段落中所記載之硫胺、甘油、CaCl 2、MgSO 4與異丙基-β-D-硫代半乳糖苷之相關說明相同，故不於此重複以避免贅述。 Similarly, the description of the concentration of thiamine, glycerin, CaCl 2 , MgSO 4 and isopropyl-β-D-thiogalactoside can also be described in the paragraph related to the method for producing indigo dye of the present invention above The related descriptions of thiamine, glycerol, CaCl 2 , MgSO 4 and isopropyl-β-D-thiogalactoside are the same, so they are not repeated here to avoid repetition.
又，於另一實施例中，上述培養基除了包含麩胺酸及/或其鹽類、M9鹽類溶液、硫胺、甘油、CaCl 2、MgSO 4與異丙基-β-D-硫代半乳糖苷之外，還可更包含一酵母菌萃取物。酵母菌萃取物於上述培養基中的濃度可為約0.05-30 g/L，例如，約0.1-30 g/L、約0.5-25 g/L、約1-20 g/L，約2-18 g/L、約3-15 g/L、約3-10 g/L、約5-20 g/L等，但不限於此。在一實施例中，酵母菌萃取物於上述培養基中的濃度可為約1 g/L、3 g/L、5 g/L或20 g/L。 Also, in another embodiment, the above-mentioned culture medium contains glutamine and/or its salts, M9 salt solution, thiamine, glycerol, CaCl 2 , MgSO 4 and isopropyl-β-D-thiosemi In addition to lactosides, a yeast extract can also be included. The concentration of the yeast extract in the above medium may be about 0.05-30 g/L, for example, about 0.1-30 g/L, about 0.5-25 g/L, about 1-20 g/L, about 2-18 g/L, about 3-15 g/L, about 3-10 g/L, about 5-20 g/L, etc., but not limited thereto. In one embodiment, the concentration of the yeast extract in the above-mentioned culture medium may be about 1 g/L, 3 g/L, 5 g/L or 20 g/L.
又，於本發明之培養基用於培養一經基因改質的微生物並使其生產靛藍染料的用途中，上述經基因改質的微生物可包括，但不限於一外源性核酸序列，其編碼萘二氧酶。In addition, in the use of the culture medium of the present invention for cultivating a genetically modified microorganism and making it produce indigo dye, the above-mentioned genetically modified microorganism may include, but is not limited to, an exogenous nucleic acid sequence encoding naphthalene Oxygenase.
上述外源性核酸序列可包括與序列辨識號：1具有至少85%之序列相似度的一序列，但不限於此。在一實施例中，上述外源性核酸序列可包括序列辨識號：1之序列。在另一實施例中，上述外源性核酸序列可為序列辨識號：1之序列。The above-mentioned exogenous nucleic acid sequence may include a sequence with at least 85% sequence similarity to sequence identification number: 1, but is not limited thereto. In one embodiment, the above-mentioned exogenous nucleic acid sequence may include a sequence of sequence identification number: 1. In another embodiment, the above-mentioned exogenous nucleic acid sequence may be a sequence with sequence identification number: 1.
再者，於一實施例中，於本發明之培養基用於培養一經基因改質的微生物並使其生產靛藍染料的用途中，關於上述經基因改質的微生物，除了可包括上述編碼萘二氧酶之外源性核酸序列之外，其之內源性 icd基因還可進一步被剔除。 Furthermore, in one embodiment, in the use of the culture medium of the present invention for cultivating a genetically modified microorganism and making it produce indigo dye, the above-mentioned genetically modified microorganism may include the above-mentioned encoding naphthalene dioxygen In addition to the exogenous nucleic acid sequence of the enzyme, the endogenous icd gene can be further deleted.
又，於一特定實施例中，於本發明之培養基用於培養一經基因改質的微生物並使其生產靛藍染料的用途中，所述之微生物可為上述任何之本發明之用於生產靛藍染料之經基因改質的微生物、上述本發明之新穎之大腸桿菌基因改質株、或藉由任何上述之本發明之製備用於生產靛藍染料之經基因改質的微生物的方法所製備出之任何經基因改質的微生物。Furthermore, in a specific embodiment, in the use of the culture medium of the present invention for cultivating a genetically modified microorganism to produce indigo dye, the microorganism may be any of the above-mentioned indigo dyes of the present invention The genetically modified microorganism, the novel E. coli genetically modified strain of the present invention described above, or any of the above-described methods of the present invention for preparing genetically modified microorganisms for the production of indigo dye Genetically modified microorganisms.
於本發明之培養基用於培養一經基因改質的微生物並使其生產靛藍染料的用途中，所述之培養基具有使微生物有效利用麩胺酸並有效提升靛藍染料產量的功效。In the application of the culture medium of the present invention for cultivating a genetically modified microorganism and making it produce indigo dye, the culture medium has the effect of enabling the microorganism to effectively utilize glutamic acid and effectively increasing the production of indigo dye.
實施例Example
1. 實施例11. Example 1
1.1 菌株之基因改質1.1 Genetic modification of strains
以大腸桿菌BW25113為基因改質之來源菌株，製備表1中所示的各菌株。Using Escherichia coli BW25113 as the source strain for genetic modification, the strains shown in Table 1 were prepared.
表1
1.1.1 大腸桿菌JDG0（經剔除內源性 icd基因的大腸桿菌BW25113）的製備 1.1.1 Preparation of Escherichia coli JDG0 (E. coli BW25113 with endogenous icd gene removed)
透過於文獻Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5.中所記載之染色體基因之一步施活(one-step inactivation of chromosome gene)方法來將大腸桿菌BW25113之內源性 icd基因剔除。此方法係藉由與於染色體上之 icd基因的序列互補的引子，透過同源序列互換(homologous recombination)，將大腸桿菌BW25113染色體上之 icd基因剔除。 Through the one-step inactivation of chromosome gene method described in the literature Proc Natl Acad Sci US A. 2000 Jun 6;97(12):6640-5. to transform E. coli BW25113 into The source icd gene was eliminated. This method is based on the icd gene sequence by the primer is complementary to the chromosome through homologous sequences swap (homologous recombination), E. coli BW25113 icd gene knockout on the chromosome.
接著，可利用聚合酶鏈鎖反應(polymerase chain reaction)來確認經剔除 icd基因之大腸桿菌BW25113。經確認染色體上沒有 icd基因的大腸桿菌BW25113，即為大腸桿菌JDG0。 Then, the polymerase chain reaction can be used to confirm the E. coli BW25113 with the icd gene deleted. It is confirmed that E. coli BW25113 without the icd gene on the chromosome is E. coli JDG0.
1.2 經馴化之大腸桿菌的製備1.2 Preparation of domesticated Escherichia coli
將所獲得之大腸桿菌JDG0，以逐步提升培養基內麩胺酸濃度(1 g/L-10 g/L)的方式進行馴化。所採用之培養基為M9 (minimum medium)培養基。M9培養基之配方如表2中所示，另於M9培養基中添加甘油10 g/L作為碳源。The obtained Escherichia coli JDG0 was acclimated by gradually increasing the concentration of glutamate in the medium (1 g/L-10 g/L). The medium used is M9 (minimum medium) medium. The formula of the M9 medium is shown in Table 2. In addition, 10 g/L of glycerol was added to the M9 medium as a carbon source.
表2
將大腸桿菌JDG0之單一菌落接入搖瓶中，並培養於37 oC。於培養72小時後，取適量菌液塗佈至含有一起始麩胺酸濃度之平板培養基上，並於37 oC培養48-72小時。採用上述起始麩胺酸濃度之培養基進行3-6個循環。之後，挑取大顆菌落進行下一回合具較高麩胺酸濃度之培養，且同樣進行3-6個循環。依上述方式逐步增加麩胺酸的濃度來培養菌株。 Put a single colony of E. coli JDG0 into a shake flask and culture it at 37 o C. After 72 hours of incubation, an appropriate amount applied to bacteria on a plate medium containing a concentration of a starting glutamic acid and cultured at 37 o C 48-72 hours. Use the above-mentioned initial glutamic acid concentration medium for 3-6 cycles. After that, pick large colonies for the next round of culture with a higher glutamic acid concentration, and also perform 3-6 cycles. Gradually increase the concentration of glutamic acid to cultivate the strains in the above manner.
於每回合不同之麩胺酸濃度的培養皆選出一菌株。所挑選出的菌株依據麩胺酸培養濃度低至高分別為，大腸桿菌JDG1、大腸桿菌JDG2、大腸桿菌JDG3與大腸桿菌JDG4。In each round of cultivation with different glutamic acid concentrations, one strain was selected. The selected strains were E. coli JDG1, E. coli JDG2, E. coli JDG3 and E. coli JDG4 according to the low to high glutamine culture concentration.
將大腸桿菌JDG0、大腸桿菌JDG1、大腸桿菌JDG2、大腸桿菌JDG3與大腸桿菌JDG4進行麩胺酸消耗速率之分析。結果如表3所示。E. coli JDG0, E. coli JDG1, E. coli JDG2, E. coli JDG3 and E. coli JDG4 were analyzed for the consumption rate of glutamate. The results are shown in Table 3.
表3
依據表3可知，大腸桿菌JDG4具有最高之消耗麩胺酸速率。According to Table 3, E. coli JDG4 has the highest consumption rate of glutamine.
1.3 萘二氧酶(naphthalene dioxygenase, NDO)表現之菌株的製備1.3 Preparation of strains showing naphthalene dioxygenase (NDO)
將可表現萘二氧酶(naphthalene dioxygenase, NDO)之基因（其來源物種為戀臭假單孢菌( Pseudomonas putida)，且其序列為序列辨識號：1之序列）的質體DNA(JDBP00)以電穿孔方式導入上方所獲得具有最高之麩胺酸消耗速率的大腸桿菌JDG4。 The plastid DNA (JDBP00) that can express the gene of naphthalene dioxygenase (NDO) (the source species is Pseudomonas putida , and its sequence is the sequence of sequence identification number: 1) The Escherichia coli JDG4 with the highest glutamate consumption rate obtained above was introduced by electroporation.
接著，藉由抗生素篩選方式得到能夠表現萘二氧酶之基因的菌株，並將所篩選出之菌株命名為大腸桿菌JD938。大腸桿菌JD938於民國107年11月22日寄存於中華民國食品工業發展研究所生物資源保存及研究中心，寄存編號為BCRC 940687。 Next, a strain capable of expressing the naphthalene dioxygenase gene was obtained by antibiotic screening, and the strain selected was named Escherichia coli JD938. Escherichia coli JD938 was deposited at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China on November 22, 1987, and the deposit number is BCRC 940687.
實施例2 Example 2
將大腸桿菌JD904以LB培養基於37℃進行隔夜培養。 Escherichia coli JD904 was cultured in LB medium at 37°C overnight.
之後，將大腸桿菌JD904接種於一實驗培養基或一比較培養基以進行醱酵槽培養。 Afterwards, E. coli JD904 was inoculated into an experimental medium or a comparative medium for fermentation tank culture.
其中，比較培養基的成分如下所述：酵母菌萃取物(yeast extract.YE)20g/L、麩胺酸10g/L、甘油50g/L、色胺酸(tryptophan)5g/L、異丙基-β-D-硫代半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside,IPTG)0.5mM、CaCl2 1μM、MgSO4 1mM、硫胺(thiamine)1mg/L與1X M9鹽類。關於實驗培養基之成分，相較於比較培養基，除了酵母菌萃取物20g/L被置換為麩胺酸10g/L，其餘成分皆與比較培養基的成分相同。 Among them, the composition of the comparison medium is as follows: yeast extract (YE) 20g/L, glutamine 10g/L, glycerol 50g/L, tryptophan 5g/L, isopropyl- β-D-thiogalactopyranoside (Isopropyl β-D-1-thiogalactopyranoside, IPTG) 0.5mM, CaCl 2 1μM, MgSO 4 1mM, thiamine (thiamine) 1mg/L and 1X M9 salt. Regarding the composition of the experimental medium, compared to the comparison medium, except that the yeast extract 20g/L was replaced with 10g/L glutamic acid, the rest of the components were the same as the comparison medium.
而醱酵槽培養條件如下所述：醱酵槽溫度30℃、pH 7.0、溶氧(dissolved oxygen,D.O.)10%。 The fermentation conditions of the fermenter are as follows: the temperature of the fermenter is 30°C, the pH is 7.0, and the dissolved oxygen (D.O.) 10%.
於培養72小時後，以分光光度計測量醱酵液之OD620數值。將所測得之OD620數值對照靛藍染料(indigo)濃度標準曲線以獲得靛藍染料濃度(靛藍染料產量)(mg/L)。 After 72 hours of incubation, the OD 620 of the fermented liquid was measured with a spectrophotometer. Compare the measured OD 620 value with the indigo concentration standard curve to obtain the indigo dye concentration (indigo dye yield) (mg/L).
另，將200mL之醱酵液離心並去除上清液以獲得沉 澱之菌塊，之後將菌塊進行冷凍乾燥並秤重以確認菌體乾重。藉由靛藍染料濃度與菌體乾重計算出菌體中之靛藍染料含量(mg/g菌體乾重)。 In addition, centrifuge 200 mL of fermented liquid and remove the supernatant to obtain a precipitate The pellets are then freeze-dried and weighed to confirm the dry weight of the bacteria. Calculate the indigo dye content (mg/g dry weight of the bacteria) in the bacteria by the indigo dye concentration and the dry weight of the bacteria.
結果顯示於第1A圖與第1B圖。 The results are shown in Figure 1A and Figure 1B.
由第1A圖與第1B圖顯示，以實驗培養基(含有麩胺酸之培養基)培養之大腸桿菌JD904的靛藍染料產量與以比較培養基(未含麩胺酸之培養基)培養之大腸桿菌JD904相近，然而，以實驗培養基(含有麩胺酸之培養基)培養之大腸桿菌JD904中之靛藍染料的含量為以比較培養基(未含麩胺酸之培養基)培養之大腸桿菌JD904中之靛藍染料的含量的約2.3倍。 Figures 1A and 1B show that the indigo dye production of E. coli JD904 cultured in experimental medium (medium containing glutamic acid) is similar to that of E. coli JD904 cultured in comparative medium (medium without glutamic acid). However, the content of indigo dye in Escherichia coli JD904 cultured on the experimental medium (medium containing glutamate) is about the amount of indigo dye in E. coli JD904 cultured on the comparative medium (medium without glutamate). 2.3 times.
由上述結果可知，藉由使用含有麩胺酸之培養基來培養大腸桿菌，可有效提升大腸桿菌生產之靛藍染料的純度。 It can be seen from the above results that the purity of the indigo dye produced by E. coli can be effectively improved by using a medium containing glutamic acid to cultivate E. coli.
實施例3 Example 3
將大腸桿菌JD904以LB培養基於37℃進行隔夜培養。 Escherichia coli JD904 was cultured in LB medium at 37°C overnight.
之後，將大腸桿菌JD904接種於一實驗培養基或一比較培養基以進行醱酵槽培養。 Afterwards, E. coli JD904 was inoculated into an experimental medium or a comparative medium for fermentation tank culture.
其中，比較培養基的成分如下所述：酵母菌萃取物20g/L、甘油50g/L、色胺酸5g/L、異丙基-β-D-硫代半乳糖苷0.5mM、CaCl2 1μM、MgSO4 1mM、硫胺1mg/L與1X M9鹽類。關於實驗培養基之成分，相較於比較培養基，除了額外添加麩胺酸10g/L以及將酵母菌萃取物調整為1g/L之外，其餘成分皆與比較培養基的成分相同。 Among them, the composition of the comparison medium is as follows: yeast extract 20g/L, glycerol 50g/L, tryptophan 5g/L, isopropyl-β-D-thiogalactoside 0.5mM, CaCl 2 1μM, MgSO 4 1mM, Thiamine 1mg/L and 1X M9 salts. Regarding the composition of the experimental medium, compared to the comparison medium, except for the addition of 10g/L of glutamic acid and the adjustment of the yeast extract to 1g/L, the other components are the same as those of the comparison medium.
而醱酵槽培養條件如下所述：醱酵槽溫度30 oC、pH 7.0、溶氧10%。 The fermentation conditions of the fermenter are as follows: the temperature of the fermenter is 30 o C, the pH is 7.0, and the dissolved oxygen is 10%.
於培養72小時後，以分光光度計測量醱酵液之OD 620數值。將所測得之OD 620數值對照靛藍染料(indigo)濃度標準曲線以獲得靛藍染料濃度(mg/L)。 After 72 hours of incubation, the OD 620 of the fermented liquid was measured with a spectrophotometer. Compare the measured OD 620 value with the indigo concentration standard curve to obtain the indigo dye concentration (mg/L).
另，將200 mL之醱酵液離心並去除上清液以獲得沉澱之菌塊，之後將菌塊進行冷凍乾燥並秤重以確認菌體乾重。藉由靛藍染料濃度與菌體乾重計算出於菌體中靛藍染料含量累積效率（mg/g菌體乾重/小時）。Separately, centrifuge 200 mL of the fermentation broth and remove the supernatant to obtain the precipitated bacterial mass, then freeze-dry the bacterial mass and weigh to confirm the dry weight of the bacterial cell. Calculate the accumulation efficiency of indigo dye content in the bacteria by the indigo dye concentration and the dry weight of the bacteria (mg/g dry weight of the bacteria/hour).
結果顯示於第2A圖與第2B圖。The results are shown in Figure 2A and Figure 2B.
由第2A圖與第2B圖顯示，以實驗培養基（含有麩胺酸之培養基）培養之大腸桿菌JD904的靛藍染料產量與以比較培養基（未含麩胺酸之培養基）培養之大腸桿菌JD904相近，然而，以實驗培養基（含有麩胺酸之培養基）培養之大腸桿菌JD904的靛藍染料含量累積效率為以比較培養基（未含麩胺酸之培養基）培養之大腸桿菌JD904的靛藍染料含量累積效率的約1.3倍。Figures 2A and 2B show that the indigo dye production of Escherichia coli JD904 cultivated in experimental medium (medium containing glutamate) is similar to that of E. coli JD904 cultivated on comparative medium (medium without glutamate). However, the cumulative efficiency of the indigo dye content of E. coli JD904 cultured on the experimental medium (medium containing glutamate) is about the same as the cumulative efficiency of the indigo dye content of E. coli JD904 cultured on the comparative medium (medium without glutamate). 1.3 times.
由上述結果可知，藉由使用含有麩胺酸及添加少量酵母菌萃取物之培養基培養，可使大腸桿菌更快速地生產靛藍染料。From the above results, it can be seen that by using a culture medium containing glutamic acid and adding a small amount of yeast extract, Escherichia coli can produce indigo dye more quickly.
實施例4Example 4
分別將大腸桿菌JD904與JD938以LB培養基於37 oC進行隔夜培養。 E. coli JD904 and JD938 were cultured in LB medium at 37 o C overnight.
之後，分別將大腸桿菌JD904與JD938接種於一實驗培養基，並於30 oC以搖瓶進行培養。 Thereafter, the E. coli were inoculated in a JD904 and JD938 experimental medium, and cultured at 30 o C in a shake flask.
實驗培養基的成分如下所述：麩胺酸10 g/L、甘油50 g/L、色胺酸5 g/L、異丙基-β-D-硫代半乳糖苷0.5 mM、CaCl 21 μM、MgSO 41 mM、硫胺1 mg/L與1X M9鹽類。 The composition of the experimental medium is as follows: glutamine 10 g/L, glycerol 50 g/L, tryptophan 5 g/L, isopropyl-β-D-thiogalactoside 0.5 mM, CaCl 2 1 μM , MgSO 4 1 mM, Thiamine 1 mg/L and 1X M9 salts.
於培養24小時後，以分光光度計測量細菌懸浮液之OD 600數值以確認菌體之生長情況。另，將1 mL之細菌懸浮液離心，並將所獲得之上清液藉由YSI生化分析儀（機型YSI 2700，慧技科學）分析其中麩胺酸的含量以推算菌體之麩胺酸消耗量。 After 24 hours of culture, the OD 600 value of the bacterial suspension was measured with a spectrophotometer to confirm the growth of the bacteria. In addition, 1 mL of the bacterial suspension was centrifuged, and the obtained supernatant was analyzed by the YSI biochemical analyzer (model YSI 2700, Smart Science) to analyze the content of glutamic acid to calculate the glutamic acid of the bacteria. consumption.
結果顯示於第3A圖與第3B圖。The results are shown in Figure 3A and Figure 3B.
第3A圖與第3B圖顯示，大腸桿菌JD938之麩胺酸消耗量約為大腸桿菌JD904的20倍，且大腸桿菌JD938之菌體濃度可為大腸桿菌JD904的約8倍。Figures 3A and 3B show that the consumption of glutamate of E. coli JD938 is about 20 times that of E. coli JD904, and the cell concentration of E. coli JD938 can be about 8 times that of E. coli JD904.
由上述結果可知，大腸桿菌JD938相較於大腸桿菌JD904可更有效地利用麩胺酸生長。From the above results, it can be seen that E. coli JD938 can use glutamine to grow more effectively than E. coli JD904.
實施例5Example 5
分別將大腸桿菌JD904與JD938以LB培養基於37 oC進行隔夜培養。 E. coli JD904 and JD938 were cultured in LB medium at 37 o C overnight.
之後，分別將大腸桿菌JD904與JD938接種於一實驗培養基，並於30 oC以搖瓶進行培養。 Thereafter, the E. coli were inoculated in a JD904 and JD938 experimental medium, and cultured at 30 o C in a shake flask.
實驗培養基的成分如下所述：麩胺酸10 g/L、甘油10 g/L、色胺酸5 g/L、異丙基-β-D-硫代半乳糖苷0.5 mM、CaCl 21 μM、MgSO 41 mM、硫胺1 mg/L與1X M9鹽類。 The composition of the experimental medium is as follows: glutamine 10 g/L, glycerol 10 g/L, tryptophan 5 g/L, isopropyl-β-D-thiogalactoside 0.5 mM, CaCl 2 1 μM , MgSO 4 1 mM, Thiamine 1 mg/L and 1X M9 salts.
於培養24小時後，將培養液經離心以取得菌體，接著加入溶劑二甲基亞碸(dimethyl sulfoxide, DMSO)混合均勻以獲得萃取液，再以分光光度計測量萃取液之OD 620數值。將所測得之OD 620數值對照靛藍染料濃度標準曲線以獲得靛藍染料濃度(mg/L)。 After culturing for 24 hours, the culture solution was centrifuged to obtain bacterial cells, and then the solvent dimethyl sulfoxide (DMSO) was added and mixed uniformly to obtain the extract, and then the OD 620 value of the extract was measured with a spectrophotometer. Compare the measured OD 620 value with the indigo dye concentration standard curve to obtain the indigo dye concentration (mg/L).
結果顯示於第4圖。The results are shown in Figure 4.
第4圖顯示，大腸桿菌JD938之靛藍染料產量為大腸桿菌JD904的3倍以上。Figure 4 shows that the indigo dye production of E. coli JD938 is more than three times that of E. coli JD904.
由上述結果可知，大腸桿菌JD938相較於大腸桿菌JD904，可更有效地利用麩胺酸生長並提高生產靛藍染料的效率。From the above results, it can be seen that compared with E. coli JD904, Escherichia coli JD938 can more effectively use glutamate to grow and improve the efficiency of indigo dye production.
實施例6Example 6
6.1 培養基中之麩胺酸濃度測試6.1 Test of Glutamic Acid Concentration in Culture Medium
分別將大腸桿菌JD904與JD938以LB培養基於37 oC進行隔夜培養。 E. coli JD904 and JD938 were cultured in LB medium at 37 o C overnight.
之後，將大腸桿菌JD904或大腸桿菌JD938接種於含不同麩胺酸濃度之實驗培養基，並於30 oC以搖瓶進行培養。 After that, E. coli JD904 or E. coli JD938 was inoculated into experimental media with different glutamic acid concentrations and cultured in shake flasks at 30 o C.
實驗培養基的成分如下所述：麩胺酸（1 g/L、5 g/L、10 g/L、15 g/L、20 g/L、30 g/L或40 g/L）、甘油(10 g/L)、色胺酸(5 g/L)、異丙基-β-D-硫代半乳糖苷(0.5 mM)、CaCl 2(1 μM)、MgSO 4(1 mM)、硫胺(1 mg/L)與1X M9鹽類。 The composition of the experimental medium is as follows: glutamine (1 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 30 g/L or 40 g/L), glycerol ( 10 g/L), tryptophan (5 g/L), isopropyl-β-D-thiogalactoside (0.5 mM), CaCl 2 (1 μM), MgSO 4 (1 mM), thiamine (1 mg/L) and 1X M9 salts.
於培養24小時後，將培養液經離心以取得菌體，接者加入溶劑二甲基亞碸混合均勻以獲得萃取液，再以分光光度計測量萃取液之OD 620數值。將所測得之OD 620數值對照靛藍染料濃度標準曲線以獲得靛藍染料濃度(mg/L)。 After 24 hours of incubation, the culture solution was centrifuged to obtain bacterial cells, and then the solvent dimethyl sulfoxide was added and mixed uniformly to obtain the extract, and then the OD 620 value of the extract was measured with a spectrophotometer. Compare the measured OD 620 value with the indigo dye concentration standard curve to obtain the indigo dye concentration (mg/L).
結果如表4所示。The results are shown in Table 4.
表4
由表4所示之結果顯示，麩胺酸濃度於約10 g/L時，大腸桿菌JD938可達到最高之靛藍染料產量。又，在不同麩胺酸濃度下，大腸桿菌JD938之靛藍染料產量皆高於大腸桿菌JD904之靛藍染料產量。根據上述結果可知，在添加少量麩胺酸之情況，即從大腸桿菌JD938獲得高產量之靛藍染料。The results shown in Table 4 show that when the glutamate concentration is about 10 g/L, E. coli JD938 can achieve the highest indigo dye production. In addition, the indigo dye production of E. coli JD938 was higher than that of E. coli JD904 under different glutamate concentrations. According to the above results, it is known that in the case of adding a small amount of glutamic acid, high yield of indigo dye can be obtained from E. coli JD938.
6.2培養基中之酵母菌萃取物之濃度測試6.2 Concentration test of yeast extract in culture medium
將大腸桿菌JD938以LB培養基於37 oC進行隔夜培養。 E. coli JD938 LB medium to be cultured overnight on 37 o C.
之後，將大腸桿菌JD938接種於含不同酵母菌萃取物濃度之實驗培養基，並於30 oC以搖瓶進行培養。 After that, E. coli JD938 was inoculated into experimental media containing different yeast extract concentrations and cultured in shake flasks at 30 o C.
實驗培養基的成分如下所述：麩胺酸 10 g/L、酵母菌萃取物（0 g/L、1 g/L、3 g/L或5 g/L）、甘油(10 g/L)、色胺酸(5 g/L)、異丙基-β-D-硫代半乳糖苷(0.5 mM)、CaCl 2(1 μM)、MgSO 4(1 mM)、硫胺(1 mg/L)與1X M9鹽類。 The components of the experimental medium are as follows: glutamine 10 g/L, yeast extract (0 g/L, 1 g/L, 3 g/L or 5 g/L), glycerol (10 g/L), Tryptophan (5 g/L), isopropyl-β-D-thiogalactoside (0.5 mM), CaCl 2 (1 μM), MgSO 4 (1 mM), thiamine (1 mg/L) With 1X M9 salt.
於培養24小時後，將培養液經離心以取得菌體，接者加入溶劑二甲基亞碸進行萃取，再以分光光度計測量萃取液之OD 620數值。將所測得之OD 620數值對照靛藍染料濃度標準曲線以獲得靛藍染料濃度(mg/L)。 After culturing for 24 hours, the culture solution was centrifuged to obtain the bacteria, and then the solvent dimethyl sulfoxide was added for extraction, and then the OD 620 value of the extract was measured with a spectrophotometer. Compare the measured OD 620 value with the indigo dye concentration standard curve to obtain the indigo dye concentration (mg/L).
結果如表5所示。The results are shown in Table 5.
表5
由表5所示之結果可知，添加少量酵母菌萃取物（濃度約1 g/L時），大腸桿菌JD938可達到最高之靛藍染料產量。From the results shown in Table 5, it can be seen that adding a small amount of yeast extract (at a concentration of about 1 g/L), E. coli JD938 can achieve the highest indigo dye production.
雖然本發明已以較佳實施例揭露如上，然其並非用以限定本發明，任何熟習此技藝者，在不脫離本發明之精神和範圍內，當可作些許之更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。Although the present invention has been disclosed in preferred embodiments as above, it is not intended to limit the present invention. Anyone familiar with the art can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the present invention The scope of protection shall be subject to the scope of the attached patent application.
無。no.
第1A圖顯示，分別以實驗培養基（含有麩胺酸之培養基）與比較培養基（未含麩胺酸之培養基）培養之大腸桿菌JD904之靛藍染料的產量。 第1B圖顯示，分別以實驗培養基（含有麩胺酸之培養基）與比較培養基（未含麩胺酸之培養基）培養之大腸桿菌JD904中之靛藍染料的含量（mg/g菌體乾重）。 第2A圖顯示，分別以實驗培養基（含有麩胺酸之培養基）與比較培養基（未含麩胺酸之培養基）培養之大腸桿菌JD904之靛藍染料的產量。 第2B圖顯示，分別以實驗培養基（含有麩胺酸之培養基）與比較培養基（未含麩胺酸之培養基）培養之大腸桿菌JD904的靛藍染料含量累積效率（mg/g菌體乾重/小時）。 第3A圖顯示，以實驗培養基（含有麩胺酸之培養基）培養之大腸桿菌JD904及大腸桿菌JD938的麩胺酸消耗量。 第3B圖顯示，以實驗培養基（含有麩胺酸之培養基）培養之大腸桿菌JD904及大腸桿菌JD938的菌株生長。 第4圖顯示，以實驗培養基（含有麩胺酸之培養基）培養之大腸桿菌JD904及大腸桿菌JD938之靛藍染料的產量。Figure 1A shows the yield of indigo dye of E. coli JD904 cultured in experimental medium (medium containing glutamate) and comparison medium (medium not containing glutamic acid). Figure 1B shows the indigo dye content (mg/g dry cell weight) of E. coli JD904 cultured with experimental medium (medium containing glutamic acid) and comparison medium (medium without glutamic acid). Figure 2A shows the yield of indigo dye of E. coli JD904 cultured with experimental medium (medium containing glutamic acid) and comparison medium (medium without glutamic acid). Figure 2B shows the cumulative efficiency of indigo dye content (mg/g dry cell weight/hour) of E. coli JD904 cultured with experimental medium (medium containing glutamic acid) and comparison medium (medium without glutamic acid) ). Figure 3A shows the consumption of glutamate of E. coli JD904 and E. coli JD938 cultured in experimental medium (medium containing glutamate). Figure 3B shows the growth of E. coli JD904 and E. coli JD938 strains cultured in experimental medium (medium containing glutamic acid). Figure 4 shows the production of indigo dye of E. coli JD904 and E. coli JD938 cultured with experimental medium (medium containing glutamic acid).
1. 大腸桿菌JD938 中華民國食品工業發展研究所生物資源保存及研究中心 民國107年11月22日 BCRC 9406871. Escherichia coli JD938 Biological Resource Conservation and Research Center, Food Industry Development Research Institute, Republic of China November 22, 1987 BCRC 940687
<110> 財團法人工業技術研究院 <110> Industrial Technology Research Institute
<120> 經基因改質的微生物與生產靛藍染料的方法 <120> Genetically modified microorganisms and methods for producing indigo dye
<160> 1 <160> 1
<170> PatentIn version 3.5 <170> PatentIn version 3.5
<210> 1 <210> 1
<211> 3464 <211> 3464
<212> DNA <212> DNA
<213> 戀臭假單孢菌 <213> Pseudomonas sp
<400> 1
無。no.
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Non-Patent Citations (3)
| Title |
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| 、Fuchao Xu etal. Efficient production of indigoidine in Escherichia coli. Fermentation, Cell Culture and Bioengineering (2015)42:1149-1155. |
| BURT D.ENSLEY etal. Expression of Naphthalene Oxidation Genes in Escherichia coli Results in the Biosynthesis of Indigo. Center for Applied Microbiology and Department of Microbiology. 15 February 1983; revised 25 April 1983. |
| BURT D.ENSLEY etal. Expression of Naphthalene Oxidation Genes in Escherichia coli Results in the Biosynthesis of Indigo. Center for Applied Microbiology and Department of Microbiology. 15 February 1983; revised 25 April 1983. 、Fuchao Xu etal. Efficient production of indigoidine in Escherichia coli. Fermentation, Cell Culture and Bioengineering (2015)42:1149-1155. * |
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