TW202026419A - Genetically modified microorganism and method for producing indigo dye - Google Patents

Abstract

A genetically modified microorganism includes: an exogenous nucleic acid sequence encoding naphthalene dioxygenase (NDO), wherein the endogenousicd gene of the genetically modified microorganism is knock out, in which the endogenousicd gene encods isocitrate dehydrogenase (IDH), and wherein the genetically modified microorganism is capable of using glutamic acid and/or a salt thereof as a nitrogen source to grow and producing indigo dye.

Description

Genetically modified microorganism and method for producing indigo dye

The present invention relates to the production of indigo dyes, and in particular to genetically modified microorganisms used for the production of indigo dyes and their preparation methods, methods for producing indigo dyes and culture medium for cultivating genetically modified microorganisms and Use it to produce indigo dye.

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 involves the conversion of precursors, such as tryptophan or indole, to indole dye through oxidative enzymes. However, the current production of indigo by biological methods It is still difficult to enter the scale of industrial production.

Gluamic acid (GA) is a widely used seasoning with a low price and sufficient sources. However, there is no strain and production method that can effectively use glutamic acid as a raw material to produce indigo dye.

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.

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 microorganism has the ability to use glutamic acid and/or its salt as a nitrogen source to grow and produce indigo.

The present invention also provides a novel genetically modified strain of Escherichia coli, its 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.

The present invention also provides a method for preparing genetically modified microorganisms, including the following steps: (a) removing the endogenous icd gene of a microorganism to obtain an icd gene-removed microorganism; (b) containing glutamine and / Or its salt medium to domesticate the microorganisms that have been knocked out of the icd gene to obtain a domesticated microorganism; and (c) introduce an exogenous nucleic acid sequence encoding naphthalene dioxygenase into the domesticated microorganism, To obtain the genetically modified microorganism.

The present invention also provides a method for producing indigo dye, which includes 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 microorganism includes the above-mentioned genetically modified microorganism, the above-mentioned novel E. coli genetically modified strain, or the above-mentioned genetically modified microorganism prepared by the method for preparing genetically modified microorganism microorganism.

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.

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:

The present invention provides a genetically modified microorganism. The genetically modified microbial system of the present invention can be used to produce indigo dye.

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.

The microbial sources of the genetically modified microorganisms of the present invention may include, but are not limited to bacteria or fungi.

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 may include Escherichia coli , but are not limited thereto. In addition, the aforementioned E. coli may include E. coli BW25113, K12, DH5a, BL21, or XL1-blue, but is not limited thereto.

However, the above-mentioned examples of the microbial-derived fungi of the genetically modified microorganisms of the present invention may include Yarrowia , Pichia , Rhodotorula , Saccharomyces ( Saccharomyces ), Dekkera ( Dekkera ), Aspergillus ( Aspergillus ), Kluyveromyces ( Kluyveromyces ), Penicillium ( Penicillium ), Smut fungus ( Utilago ) Not limited to this.

In one embodiment, the microorganism source of the genetically modified microorganism of the present invention may be Escherichia coli, such as Escherichia coli BW25113.

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.

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 a sequence with sequence identification number: 1.

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 the use of glutamate as a nitrogen source by microorganisms. effectiveness. Thus, the genetically modified microorganism of the present invention may comprise in addition than the exogenous nucleic acid encoding a naphthalene dioxygenase sequences other than its endogenous icd gene can also be further removed to further promote the efficiency of microbial glutamic acid .

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 Institute of the Republic of China on November 22, 1987, and the deposit number is BCRC 940687. Genetically modified plant JD938.

The present invention also provides a novel genetically modified strain of Escherichia coli, which was deposited in the Bioresource 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 JD938.

The present invention also provides a method for preparing a genetically modified microorganism, which may include the following steps, but is not limited thereto.

Firstly, the endogenous icd gene of a microorganism is deleted to obtain an icd gene deleted microorganism.

There are no special restrictions on the method of 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 carried out 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.

The aforementioned microorganisms may include, but are not limited to bacteria or fungi.

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.

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.

Then, 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 Microorganisms with icd gene knockout. 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.

In one embodiment, the above-mentioned medium containing glutamine and/or its salt may further include an M9 salt solution.

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.

In another embodiment, the above-mentioned 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 above-mentioned carbon source is glycerol, and in the above-mentioned 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.

In an 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, culture 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 culture medium 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 to this. In one embodiment, in the above-mentioned medium containing glutamate and/or its salt, the concentration of glutamate 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, but not limited to this. In addition, the above-mentioned first concentration culture depends on the 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.

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., to the stable growth period, but not limited to this.

Furthermore, in the first concentration culture, the temperature used for culturing microorganisms also depends on 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.

In another embodiment, the above-mentioned domestication of the above-mentioned icd gene knocked- out microorganisms with a medium containing glutamic acid and/or its salts may include, but is not limited to, in addition to the above-mentioned first concentration culture, After the above-mentioned first concentration 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 at the concentration of glutamic acid is cultivated to the stable growth period. In addition, the concentration of glutamic acid and/or its salt in the medium used for each round of increasing concentration culture is higher than that of the previous round of increasing concentration culture.

In addition, the number of the above-mentioned at least one round of increased concentration culture is not particularly limited, and 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 expectations, 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 thereto.

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.

In addition, in the above method of preparing a genetically modified microorganism for producing indigo dye, the above-mentioned icd gene knocked- out microorganism is domesticated in a medium containing glutamic acid and/or its salt to obtain a domesticated After the microorganism, an exogenous nucleic acid sequence encoding a naphthalene dioxygenase is introduced into the above-mentioned domesticated microorganism to obtain a genetically modified microorganism for the production of indigo dye.

The manner 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 may be determined according to the type of carrier suitable for the microorganism actually used. For example, a specific plasmid may 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 above-mentioned exogenous nucleic acid sequence can be introduced into the above-mentioned domesticated microorganism via a plastid.

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.

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.

In addition, based on 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.

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 it is not limited thereto.

Furthermore, the present invention can also provide a method for producing indigo dye.

The method for producing indigo dye of the present invention may include the following steps, but is not limited thereto.

In the method for producing indigo dye of the present invention, a genetically modified microorganism containing an exogenous nucleic acid sequence encoding a naphthalene dioxygenase can be cultured on a precursor containing glutamic acid and/or its salt and indigo dye 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.

The above examples of a genetically modified microorganism containing an exogenous nucleic acid sequence encoding 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.

In addition, in the above-mentioned medium containing glutamic acid and/or its salts and indigo dye precursor, 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, 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.

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 above-mentioned indigo dye precursor may be tryptophan, and the concentration of tryptophan may be about 0.1-25 in the medium containing glutamine and/or its salt and the indigo dye precursor. 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.

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 can be about 1 g/L, 3 g/L, 5 g/L, 20 g/L or 30 g/L.

In another embodiment, the above-mentioned culture medium containing glutamine and/or its salt and indigo dye precursor may further comprise M9 salt solution, thiamine, glycerol, CaCl ₂ , MgSO ₄ and isopropyl -β-D-thiogalactopyranoside (Isopropyl β-D-1-thiogalactopyranoside, IPTG).

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 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.

In addition, the concentration of thiamine in the medium containing glutamate and/or its salt and indigo dye precursor can 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 aforementioned medium containing glutamic acid and/or its salt and indigo dye precursor 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. 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 may 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 can 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.

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, glycerol, 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 medium containing glutamic acid and/or its salt and indigo dye precursor 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 or 20 g/L.

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 there is no particular limitation. 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., to the stable growth period, but not limited to this.

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 is not particularly limited. For example, in the production method of the present invention, an indigo dye, a microorganism can 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.

By any of the above-mentioned methods of the present invention for producing indigo dye, 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.

The invention also provides the use of a culture medium for cultivating a genetically modified microorganism and making it produce indigo dye.

The aforementioned 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 0.05-60 g/L, for example, about 0.1-60 g/L, about 0.5-55 g/L, about 0.5-40 g/L 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, the concentration of glutamic acid and/or its salts in the above medium may be about 1 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 30 g/L, 40 g/L or 50 g/L.

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, 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 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.

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 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, 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.

Furthermore, in one embodiment, the above-mentioned culture medium may further include a yeast extract in addition to glutamic acid 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.

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.

The description of the M9 salt solution and its components may 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.

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.

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, it can also contain a yeast extract. 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.

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.

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.

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.

Furthermore, in a specific embodiment, in the use of the culture medium of the present invention for culturing a genetically modified microorganism and making it 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 A genetically modified microorganism.

In the use 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.

Example

1. Example 1

1.1 Genetic modification of strains

Using Escherichia coli BW25113 as the source strain for genetic modification, the strains shown in Table 1 were prepared.

Table 1

1.1.1 Preparation of Escherichia coli JDG0 (E. coli BW25113 with endogenous icd gene removed)

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.

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 Preparation of domesticated Escherichia coli

The obtained Escherichia coli JDG0 was domesticated 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.

Table 2

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 glutamate concentration, and also perform 3-6 cycles. Gradually increase the concentration of glutamine to cultivate the strains in the above manner.

In each round of cultivation with different glutamic acid concentrations, one strain was selected. The selected strains were as follows: Escherichia coli JDG1, Escherichia coli JDG2, Escherichia coli JDG3, and Escherichia coli JDG4 according to the low to high glutamine culture concentration.

E. coli JDG0, E. coli JDG1, E. coli JDG2, E. coli JDG3, and E. coli JDG4 were analyzed for glutamine consumption rate. The results are shown in Table 3.

table 3

According to Table 3, E. coli JDG4 has the highest consumption rate of glutamine.

1.3 Preparation of strains showing naphthalene dioxygenase (NDO)

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 glutamine consumption rate obtained above was introduced by electroporation.

Then, 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.

Example 2

E. coli JD938 LB medium to be cultured overnight on 37 ^o C.

Afterwards, E. coli JD938 was inoculated into an experimental medium or a comparative medium for fermentation tank culture.

Among them, the components of the comparison medium are as follows: yeast extract (YE) 20g/L, glutamine 10 g/L, glycerol 50 g/L, tryptophan 5 g/L, iso Isopropyl β-D-1-thiogalactopyranoside (IPTG) 0.5 mM, CaCl ₂ 1 μM, MgSO ₄ 1 mM, thiamine 1 mg/L, and 1X M9 salt class. Regarding the composition of the experimental medium, compared to the comparative medium, except that 20 g/L of yeast extract is replaced with 10 g/L of glutamic acid, the rest of the components are the same as those of the comparative medium.

The culture conditions of the fermenter are as follows: the temperature of the fermenter is 30 ^o C, pH 7.0, and dissolved oxygen (DO) 10%.

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).

Separately, centrifuge 200 mL of fermented liquid and remove the supernatant to obtain precipitated bacterial clumps, then freeze-dry the bacterial clumps and weigh them 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.

The results are shown in Figure 1A and Figure 1B.

Figures 1A and 1B show that the indigo dye production of Escherichia coli JD904 cultured on the experimental medium (medium containing glutamic acid) is similar to that of E. coli JD904 cultivated on the comparative medium (medium without glutamic acid). However, the content of indigo dye in Escherichia coli JD904 cultured on the experimental medium (medium containing glutamic acid) is about the amount of indigo dye in E. coli JD904 cultured on the comparative medium (medium without glutamic acid). 2.3 times.

From the above results, it can be seen that by using a medium containing glutamic acid to cultivate E. coli, the purity of the indigo dye produced by E. coli can be effectively improved.

Example 3

E. coli JD938 LB medium to be cultured overnight on 37 ^o C.

Afterwards, E. coli JD938 was inoculated into an experimental medium or a comparative medium for fermentation tank culture.

Among them, the composition of the comparison medium is as follows: yeast extract 20 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. Regarding the composition of the experimental medium, compared to the comparison medium, except for the addition of 10 g/L of glutamic acid and the adjustment of the yeast extract to 1 g/L, the other components are the same as those of the comparison medium.

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%.

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 dye (indigo) concentration standard curve to obtain the indigo dye concentration (mg/L).

Separately, centrifuge 200 mL of fermented liquid and remove the supernatant to obtain precipitated bacterial clumps, then freeze-dry the bacterial clumps and weigh them to confirm the dry weight of the bacteria. 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).

The results are shown in Figure 2A and Figure 2B.

Figures 2A and 2B show that the indigo dye production of Escherichia coli JD904 cultured on experimental medium (medium containing glutamic acid) is similar to that of E. coli JD904 cultivated on comparative medium (medium without glutamic acid). 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 that of the indigo dye content cumulative efficiency of E. 1.3 times.

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.

Example 4

E. coli JD904 and JD938 were cultured in LB medium at 37 ^o C overnight.

Thereafter, the E. coli were inoculated in a JD904 and JD938 experimental medium, and cultured at 30 ^o C in a shake flask.

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.

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.

The results are shown in Figure 3A and Figure 3B.

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.

From the above results, it can be seen that E. coli JD938 can use glutamine to grow more effectively than E. coli JD904.

Example 5

E. coli JD904 and JD938 were cultured in LB medium at 37 ^o C overnight.

Thereafter, the E. coli were inoculated in a JD904 and JD938 experimental medium, and cultured at 30 ^o C in a shake flask.

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.

After culturing for 24 hours, the culture solution was centrifuged to obtain the bacteria, 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).

The results are shown in Figure 4.

Figure 4 shows that the indigo dye production of E. coli JD938 is more than three times that of E. coli JD904.

From the above results, it can be seen that compared with E. coli JD904, E. coli JD938 can more effectively use glutamic acid to grow and increase the efficiency of indigo dye production.

Example 6

6.1 Glutamine concentration test in the culture medium

E. coli JD904 and JD938 were cultured in LB medium at 37 ^o C overnight.

After that, E. coli JD904 or E. coli JD938 was inoculated into experimental media with different glutamine concentrations, and cultured in shake flasks at 30 ^o C.

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.

After culturing for 24 hours, 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).

The results are shown in Table 4.

Table 4

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 at different glutamate concentrations. According to the above results, it is known that in the case of adding a small amount of glutamic acid, a high yield of indigo dye can be obtained from E. coli JD938.

6.2 Concentration test of yeast extract in culture medium

E. coli JD938 LB medium to be cultured overnight on 37 ^o C.

Thereafter, Escherichia coli was inoculated into experimental medium JD938 different yeast extract concentrations contained, and cultured at 30 ^o C in a shake flask.

The composition of the experimental medium is 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.

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).

The results are shown in Table 5.

table 5

According to the results shown in Table 5, 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.

Although the present invention has been disclosed as above in the preferred embodiment, 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.

no.

Figure 1A shows the production of indigo dye of E. coli JD904 cultured in experimental medium (medium containing glutamate) and comparison medium (medium without glutamic acid). Figure 1B shows the indigo dye content (mg/g dry cell weight) of E. coli JD904 cultured with experimental medium (medium containing glutamate) 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 Escherichia 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 Escherichia 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 on experimental medium (medium containing glutamic acid).

1. Escherichia coli JD938 Biological Resources Conservation and Research Center, Food Industry Development Research Institute, Republic of China November 22, 2007 BCRC 940687

<110> Institute of Industrial Technology <120> Genetically modified microorganisms and methods for producing indigo dye <130> 0954-A26082TW<160> 1 <170> PatentIn version 3.5<210> 1<211> 3464<212 > DNA <213> Love smell Pseudomonas <400> 1atggaacttc tcatacagcc aaacaatcgc ataattccct tcagtgccgg tgccaacctt 60ctggaagtgc ttcgcgagaa cggtgtagct atttcctaca gttgcttgtc tgggcgttgc 120ggaacctgtc gctgccgggt tatagatggc agtgtcattg attctggggc ggaaaatggg 180caatcaaacc tcaccgacaa gcagtatgtg ctcgcctgtc agtcagtact cactggcaat 240tgcgctatcg aagtcccaga agccgacgaa attgtcactc acccggcgcg aatcatcaag 300ggcacagtgg tcgcagtcga gtcgcccact cacgatatcc gtcgcttacg cgtacgcctc 360tccaagccct tcgagttctc acccggacag tacgcgacac tgcagttcag ccctgagcat 420gcgcgtccgt attcaatggc aggtttgcca gatgaccaag aaatggagtt ccacatacgc 480aaggtgccgg gtgggcgcgt cacggagtat gttttcgaac acgtccgcga aggtacaagc 540atcaagttga gcgggcctct tggtacggct tatctacgtc agaagcacac cggaccgatg 600ctgtgtgtag gtggcgggac cggactcgca ccggtgctgt cgattgttcg cggcgcgctg 660aagtcgggta tgacgaaccc catcctcctt tatttcgggg tgcgcagtca gcaagacctc 720tacgacgcag agcgattgca caaactcgcc gctgaccacc ctcaactgac cgtacacacg 780gtgattgcaa cgggcccgat taatgagggt cagcgagccg gcctaattac cgatgtgatc 840gaaaaagaca tcctttcgct ggctgggtgg agggcctacc tgtgcggcgc accagcgatg 900gttgaagcgt tgtgcaccgt caccaagcat cttggaatat cacccgaaca tatttatgcc 960gatgccttct atcccggtgg gatctgaata gttcccggcc atgcacctct gtccatcgag 1020aattcatcag gaagacattc aaatgaacgt aaacaataag ggcagcgtct gtatttgcgg 1080cagcgaaatg ctccctaaat tcctcattta ccccatctga ggattgcttt atgacagtaa 1140agtggattga agcagtcgct ctttctgaca tccttgaagg tgacgtcctc ggcgtgactg 1200tcgagggcaa ggagctggcg ctgtatgaag ttgaaggcga aatctacgct accgacaacc 1260tgtgcacgca tggttccgcc cgcatgagtg atggttatct cgagggtaga gaaatcgaat 1320gccccttgca tcaaggtcgg tttgacgttt gcacaggcaa agccctgtgc gcacccgtga 1380cacagaacat caaaacatat ccagtcaaga ttgagaacct gcgcgtaatg attgatttga 1440gctaagaatt ttaacaggag gcaccccggg ccctagagcg taatcacccc cattccatct 1500tttttaggtg aaaacatgaa ttacaataat aaaatcttgg taagtgaatc tggtctgagc 1560caaaagcacc tgattcatgg cgatgaagaa cttttccaac atgaactgaa a accattttt 1620gcgcggaact ggctttttct cactcatgat agcctgattc ctgcccccgg cgactatgtt 1680accgcaaaaa tggggattga cgaggtcatc gtctcccggc agaacgacgg ttcgattcgt 1740gcttttctga acgtttgccg gcatcgtggc aagacgctgg tgagcgtgga agccggcaat 1800gccaaaggtt ttgtttgcag ctatcacggc tggggcttcg gctccaacgg tgaactgcag 1860agcgttccat ttgaaaaaga tctgtacggc gagtcgctca ataaaaaatg tctggggttg 1920aaagaagtcg ctcgcgtgga gagcttccat ggcttcatct acggttgctt cgaccaggag 1980gcccctcctc ttatggacta tctgggtgac gctgcttggt acctggaacc tatgttcaag 2040cattccggcg gtttagaact ggtcggtcct ccaggcaagg ttgtgatcaa ggccaactgg 2100aaggcacccg cggaaaactt tgtgggagat gcataccacg tgggttggac gcacgcgtct 2160tcgcttcgct cgggggagtc tatcttctcg tcgctcgctg gcaatgcggc gctaccacct 2220gaaggcgcag gcttgcaaat gacctccaaa tacggcagcg gcatgggtgt gttgtgggac 2280ggatattcag gtgtgcatag cgcagacttg gttccggaat tgatggcatt cggaggcgca 2340aagcaggaaa ggctgaacaa agaaattggc gatgttcgcg ctcggattta tcgcagccac 2400ctcaactgca ccgttttccc gaacaacagc atgctgacct gctcgggtgt tttcaaagta 2460tggaac ccga tcgacgcaaa caccaccgag gtctggacct acgccattgt cgaaaaagac 2520atgcctgagg atctcaagcg ccgcttggcc gactctgttc agcgaacgtt cgggcctgct 2580ggcttctggg aaagcgacga caatgacaat atggaaacag cttcgcaaaa cggcaagaaa 2640tatcaatcaa gagatagtga tctgctttca aaccttggtt tcggtgagga cgtatacggc 2700gacgcggtct atccaggcgt cgtcggcaaa tcggcgatcg gcgagaccag ttatcgtggt 2760ttctaccggg cttaccaggc acacgtcagc agctccaact gggctgagtt cgagcatgcc 2820tctagtactt ggcatactga acttacgaag actactgatc gctaacagac gagtcgacca 2880tgatgatcaa tattcaagaa gacaagctgg tttccgccca cgacgccgaa gagattcttc 2940gtttcttcaa ttgccacgac tctgctttgc aacaagaagc cactacgctg ctgacccagg 3000aagcgcattt gttggacatt caggcttacc gtgcttggtt agagcactgc gtggggtcag 3060aggtgcaata tcaggtcatt tcacgcgaac tgcgcgcagc ttcagagcgt cgttataagc 3120tcaatgaagc catgaacgtt tacaacgaaa attttcagca actgaaagtt cgagttgagc 3180atcaactgga tccgcaaaac tggggcaaca gcccgaagct gcgctttact cgctttatca 3240ccaacgtcca ggccgcaatg gacgtaaatg acaaagagct acttcacatc cgctccaacg 3300tcattctgca ccgggcacga cgtg gcaatc aggtcgatgt cttctacgcc gcccgggaag 3360ataaatggaa acgtggcgaa ggtggagtac gaaaattggt ccagcgattc gtcgattacc 3420cagagcgcat acttcagacg cacaatctga tggtctttct gtga 3464

Claims (26)

A genetically modified microorganism comprising: an exogenous nucleic acid sequence encoding naphthalene dioxygenase (NDO); wherein the endogenous icd gene of the genetically modified microorganism is deleted, and the internal The original icd gene encodes isocitrate dehydrogenase (IDH), and the genetically modified microorganism has the ability to use glutamic acid and/or its salt as a nitrogen source to grow and produce indigo dye (indigo ) Of the ability. The genetically modified microorganism as described in item 1 of the scope of patent application, wherein the source of the genetically modified microorganism includes bacteria or fungi. Microorganisms such as genetically modified application of paragraph 2 of the scope of the patent, wherein the bacteria include the genus Izod (Escherichia), Enterobacter (Enterobacter), Enterococcus (of Enterococcus), Lactobacillus (Lactobacillus), Lactococcus ( Lactococcus ), Pseudomonas ( Pseudomonas ), Citrobacter ( Citrobacter ), Corynebacterium ( Corynebacterium ), Evans ( Erwinia ), Klebsiella ( Klebsiella ), Morganella ( Morganella ), Pantoea ( Pantoea ), Pectobacterium ( Pectobacterium ), Proteus ( Proteus ), Salmonella ( Salmonella ), Serratia ( Serratia ) or Shigella ( Shigella ) Of bacteria. The genetically modified microorganism described in item 2 of the scope of patent application, wherein the fungus includes Yarrowia , Pichia , Rhodotorula , Saccharomyces Saccharomyces), the genus Dekkera (Dekkera), the genus aspergillus (Aspergillus), Kluyveromyces (Kluyveromyces), Penicillium (Penicillium) or the genus smut (Ustilago). The genetically modified microorganism as described in item 1 of the scope of patent application, wherein the exogenous nucleic acid sequence includes a sequence with sequence identification number: 1 having a sequence similarity of at least 85%. The genetically modified microorganism as described in item 1 of the scope of patent application, wherein the genetically modified microorganism is genetically modified BW25113, and its deposit number is BCRC 940687. A novel genetically modified strain of Escherichia coli, its deposit number is BCRC 940687, in which the endogenous icd gene of the novel genetically modified strain of Escherichia coli has been deleted and includes an exogenous nucleic acid sequence encoding naphthalene dioxygenase . A method for preparing genetically modified microorganisms, including the following steps: (a) Eliminating the endogenous icd gene of a microorganism to obtain an icd gene-eliminated microorganism; (b) Using glutamine and/or its salt The type of culture medium undergoes domestication of the icd gene knocked out microorganism to obtain a domesticated microorganism; and (c) introducing an exogenous nucleic acid sequence encoding naphthalene dioxygenase into the domesticated microorganism to obtain the domesticated microorganism Genetically modified microorganisms. The method for a genetically modified microorganism as described in item 8 of the scope of patent application, wherein the microorganism is used to produce indigo dye. The method for genetically modified microorganisms as described in item 8 of the scope of patent application, wherein the microorganisms include bacteria or fungi. The method for genetically modified microorganisms as described in item 8 of the scope of patent application, wherein the exogenous nucleic acid sequence includes a sequence with at least 85% sequence similarity with sequence identification number: 1. The method for genetically modified microorganisms as described in item 8 of the scope of the patent application, wherein the domestication includes: culturing at a first concentration, and in the first concentration culturing, the microorganisms with the icd gene knocked out contain A culture medium with a first concentration of glutamate and/or its salts is cultured to a stable growth period, and the first concentration is 0.05-80 g/L. The method for genetically modified microorganisms as described in item 12 of the scope of patent application, wherein the first concentration culture is performed for 3-6 cycles. The method for genetically modified microorganisms as described in item 12 of the scope of patent application, wherein the microorganisms whose icd gene has been knocked out are cultured for 12-84 hours to a stable growth period. The method for genetically modified microorganisms as described in item 12 of the scope of patent application, wherein the domestication further comprises: after the first concentration culture, at least one round of increased concentration culture of the icd gene knocked out microorganisms, In each round of increasing concentration culture, the icd gene knocked- out microorganism is cultured in a medium containing a concentration of glutamic acid and/or its salt higher than the first concentration to a stable growth period, and each round The concentration of glutamic acid and/or its salts in the medium used for the increased concentration culture is higher than the increased concentration of the previous round. The method for genetically modified microorganisms as described in item 15 of the scope of patent application, wherein each round of increasing concentration cultivation is carried out for about 3-6 cycles. The method for genetically modified microorganisms described in item 8 of the scope of patent application, wherein the genetically modified microorganism is a genetically modified Escherichia coli, and its deposit number is BCRC 940687. A method for producing indigo dye, comprising the following steps: (a) providing a genetically modified microorganism; and (b) cultivating the genetically modified microorganism on a glutamic acid and/or its salt and an indigo One of the dye precursors in a culture medium to produce indigo dye, wherein the genetically modified microorganism includes the genetically modified microorganism as described in item 1 of the scope of patent application, and the microorganism described in item 7 of the scope of patent application Novel genetically modified strains of Escherichia coli, or genetically modified microorganisms prepared by the method for preparing genetically modified microorganisms as described in item 8 of the scope of patent application. The method for producing indigo dye described in item 18 of the scope of patent application, wherein in the medium, the concentration of the indigo dye precursor is 0.1-25 g/L. The method for producing indigo dye as described in item 18 of the scope of patent application, wherein in the medium, the concentration of the glutamic acid and/or its salt is 0.05-80 g/L. The method for producing indigo dye as described in item 18 of the scope of patent application, wherein the medium further contains a yeast extract (YE). The method for producing indigo dye as described in item 21 of the scope of patent application, wherein the concentration of the yeast extract is 0.05-30 g/L. A medium is used for cultivating a genetically modified microorganism to produce indigo dye, wherein the medium includes a glutamic acid and/or its salt, and in the medium, the glutamic acid and/or its salt The concentration of the species is 0.05-80 g/L, and the genetically modified microorganism includes an exogenous nucleic acid sequence, which encodes a naphthalene dioxygenase. The medium described in item 23 of the scope of patent application is used for cultivating genetically modified microorganisms to produce indigo dye, wherein the medium further contains thiamine, glycerol, CaCl ₂ , MgSO ₄ and isopropyl-β -D-thiogalactoside. The medium described in item 23 of the scope of patent application is used for cultivating genetically modified microorganisms to produce indigo dye, wherein the medium also contains a yeast extract, and the concentration of the yeast extract is 0.05 -30 g/L. The use of the culture medium described in item 23 of the scope of patent application for culturing genetically modified microorganisms to produce indigo dye, wherein the genetically modified microorganism is the genetically modified microorganism as described in item 1 of the scope of patent application A modified microorganism, such as the novel E. coli genetically modified strain described in item 7 of the patent application, or a genetically modified microorganism prepared by the method of the genetically modified microorganism described in item 8 of the patent application Modified microorganisms.

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