TW201700730A - A microorganism for producing carotenoids and uses thereof - Google Patents

Abstract

The present invention provides a microorganism, comprising a vector containing carotenoids biosynthetic genes and the mutant thereof, to obtain more amounts of carotenoids.

Description

Microorganisms for producing carotenoids and their applications

The present invention relates to a microorganism capable of producing carotenoids, in particular, the microorganism comprises an improved carotenoid synthesis gene, which allows the microorganism to incrementally produce carotenoids.

Lycopene is a carotenoids with a molecular formula of C ₄₀ H ₅₆ . In nature, lycopene is widely found in red-orange fruits and vegetables such as tomatoes, grapefruits, watermelons, and papayas. Lycopene is also present in human plasma and tissues, and its proportion is greater than that of β-carotene, indicating its importance in body defense. Recently, lycopene has received great attention due to its excellent antioxidant power and physiological action, and has been developed into commercial value such as nutritional products, health care products, cosmetics, and medicines.

Because lycopene is located in the cell wall of tomato, it is not easily absorbed by the body. Therefore, even if fresh tomatoes are directly consumed, it is not easy to take a sufficient dose of lycopene. The main preparation methods of lycopene are chemical synthesis, extraction from plants (such as fresh tomatoes), production by fungi (such as Blakeslea trispora ), and production by bacteria. Among them, plant extraction is the largest, but the extraction process must overcome the obstacles of the cell wall, use various methods to break the wall and increase the yield, so the project is more complicated, the time course is longer, or the extraction cost is more expensive.

In all extraction methods, whether the extraction source is plant, fungus, or bacteria, the organism itself is extracted with an organic solvent, a supercritical fluid, a vegetable oil, etc., and it is known that it is not currently in the medium and outside the cell. , directly obtain the case of lycopene. In the prior art (such as Taiwan Patent No.: I4670182), it has been revealed that the photosynthetic bacteria have the function of producing astaxanthin and the asbestos are collected outside the bacteria after being transferred into a photosynthetic bacteria by the carrier of the synthetic astaxanthin gene. Methods. The method provides a simpler method for preparing carotenoids, which can solve the extraction time and cost problems.

The pathway for the production of lycopene in microorganisms mainly involves three enzymes: geranylgeranyl pyrophosphate synthase (GGPP synthase; Crt E) and phytoene synthase (Crt B). And phytoene desaturase (Crt I). Crt E converts farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP) by isoprenyl pyrophosphate (IPP). Next, Crt B converts GGPP to phytoene, and finally, lycopene is formed via the action of Crt I (as shown in Figure 1). Previously, it has been pointed out that by combining the CrtE, CrtB and CrtI genes of six different microorganisms, they are selected into expression vectors and expressed in E. coli, and different degrees of lycopene production are found (Applied and Environmental Microbiology (2013). ), 79(2): 610-618). In addition, based on lycopene, it can further penetrate various synthetic enzymes (such as lycopene cyclase (Crt Y), β-carotene hydroxylase (Crt Z), 2,2'-β hydroxylase ( Crt G), β-carotene ketolase (Crt W) and zeaxanthin transsaccharase (Crt X), etc., are converted into various carotenoids (as shown in Figure 2).

However, how to increase the yield of lycopene or its related carotenoids in plants or fungi more effectively is a problem that the relevant industries are currently trying to solve.

In the present invention, the use of a modified carotenoid (such as lycopene) synthetic gene (such as Crt E enzyme gene and / or Crt I enzyme gene) is transferred into a microorganism (such as the red genus), which can promote The microbial production produces carotenoids, and the carotenoids produced by the improved carotenoid synthesis gene are more produced than the simple transfer of the wild type carotenoid synthesis gene. The carotenoids have the same composition as the general carotenoids, so there is no safety concern.

In addition, the present invention also proves that after the improved carotenoid (such as lycopene) synthetic gene is transferred into a photosynthetic bacterium, the photosynthetic bacterium can be cultured (for example, cultured in a liquid medium). The produced carotenoids are secreted outside the cells. The general carotenoid (including lycopene) is fat-soluble and not easily soluble in water, so it means that the carotenoid secreted by the genetically modified photosynthetic bacteria is a water-soluble carotenoid, and further confirmed that the water is soluble. The carotenoid can be combined with a metal ion (such as calcium ion) to form an agglutination reaction, thereby precipitating the water-soluble carotenoid mixture; and the carotenoid extracted from the water-soluble carotenoid and the general carotenoid The ingredients are the same.

The terms "a", "an" and "the" are used to mean "one or more" as used herein. This description is to be construed as inclusive of the singular

The term "or" in this document means "and/or".

In the present invention, the wild type Crt E enzyme comprises an amino acid sequence of SEQ ID NO: 16, which can be encoded by a gene sequence comprising SEQ ID NO: 7. The modified or mutated Crt E enzyme comprises an amino acid sequence of SEQ ID NO: 19, which can be encoded by a gene sequence comprising SEQ ID NO: 14.

In the present invention, the wild type Crt B enzyme comprises an amino acid sequence of SEQ ID NO: 17, which can be encoded by a gene sequence comprising SEQ ID NO: 8.

In the present invention, the wild type Crt I enzyme comprises an amino acid sequence of SEQ ID NO: 18, which can be encoded by a gene sequence comprising SEQ ID NO: 9. The modified or mutated Crt I enzyme comprises an amino acid sequence of SEQ ID NO: 20, which can be encoded by a gene sequence comprising SEQ ID NO: 15.

Furthermore, the term "amino acid sequence" as used herein may further comprise functional equivalents and functional derivatives of the sequence. According to the invention, "functional equivalent" also means, in particular, at least one sequence position of the above amino acid sequence, having an amino acid different from the amino acid specifically mentioned, but the properties are still unmodified initially The same mutant as the peptide. "Functional equivalents" thus include mutants obtainable by addition, substitution, deletion and/or inversion of one or more amino acids, which may occur at any sequence position as long as they are produced according to the invention A mutant of the nature profile is sufficient. More specifically, functional equivalents exist even if the reaction pattern between the mutant and the unmodified polypeptide corresponds in nature. The "functional equivalent" in the above sense is also the "precursor" of the multi-peptide and the "functional derivative" of the multi-peptide. This article A "precursor" is a natural or synthetic precursor of such multi-peptides with or without the desired biological activity.

The present invention provides a genetically modified Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 19.

The present invention provides a genetically modified Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20.

The present invention provides a vector comprising an enzyme gene required for synthesizing a carotenoid, wherein the enzyme gene required for the synthesis of the carotenoid comprises a Crt E enzyme gene, a Crt B enzyme gene and a Crt I enzyme gene, wherein the Crt The E-enzyme gene encodes a Crt E enzyme comprising SEQ ID NO: 16 or SEQ ID NO: 19, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising SEQ ID NO: 18 or SEQ ID NO: 20.

This vector can be transferred to microorganisms. The carrier may be a circular or integrated plastid depending on the application and the application. Recombinant nucleic acid constructs or gene constructs are typically expressed in a suitable host organism and are preferably inserted into a host-specific vector which enables the gene to be optimally expressed in the host. Carriers are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P. H. et al., Edsevier, Amsterdam-New York-Oxford, 1985). In addition to the plastid, the vector also includes any other vectors known to those skilled in the art, such as phage, viruses (such as SV40, CMV, baculovirus and adenovirus), transposons, IS elements, plastids, Viscosity and linear or circular DNA. Such vectors can be autonomously replicated in a host organism or on a chromosome. In a preferred embodiment, the vector is a pET23-a vector or a pBMTB-4 vector.

The present invention provides a recombinant vector comprising a Crt I enzyme gene, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20. In a preferred embodiment, the Crt I enzyme gene comprises a gene sequence of SEQ ID NO: 15.

In a specific embodiment, the recombinant vector further comprises a Crt E enzyme gene and a Crt B enzyme gene. In a preferred embodiment, the Crt E enzyme gene encodes a Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 16 or 19. In a more preferred embodiment, the Crt E enzyme gene encodes a Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 19. In another specific embodiment, the Crt E enzyme gene comprises a gene sequence of SEQ ID NO: 7 or 14. In a preferred embodiment, the Crt E enzyme gene comprises a gene sequence of SEQ ID NO: 14.

In a specific embodiment, the Crt B enzyme gene encodes a Crt B enzyme comprising an amino acid sequence of SEQ ID NO: 17. In a preferred embodiment, the Crt B enzyme gene comprises a gene sequence of SEQ ID NO: 8. The Crt B enzyme used in the present invention is a normal or wild type Crt B enzyme.

The term "gene" as used herein refers to a segment of DNA involved in the production of a functional RNA. The gene alone comprises a coding region and a non-coding region located in front of the coding region (5' UTR) and after the coding region (3' UTR) or a combination thereof. A functional RNA can be an mRNA that is translated into a peptide, polypeptide or protein. Functional RNA can also be non-coding RNA that is not translated into a proteinaceous material but has other physiological functions. Examples of non-coding RNAs include, but are not limited to, transfer RNA (tRNA), ribosomal RNA (rRNA), small RNA, and ribozyme. "gene" can contain coding sequences (table Insertive non-coding sequence (insertion sequence) between sequences. "Coding region" or "coding sequence" means a portion of a gene that is transcribed into mRNA, and mRNA is translated into a polypeptide via a three-base codon and the initiation and termination signals for translation of the polypeptide. "Coding region" or "coding sequence" also refers to a portion of a gene that is transcribed into a non-coding but functional RNA.

In another specific embodiment, the recombinant vector further comprises a control sequence. The control sequence may be any promoter suitable for expression of the recombinant gene of the invention, such as, but not limited to, the 35S promoter and the ubiquitin promoter.

According to the present invention, the aforementioned recombinant vector further contains a drug resistance gene in addition to the above control sequence, and the drug resistance gene is, for example, but not limited to, a drug resistance gene such as kanamycin of the npt II gene.

The present invention provides a recombinant vector comprising a Crt E enzyme gene, wherein the Crt E enzyme gene encodes a Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 19. In a preferred embodiment, the Crt E enzyme gene comprises a gene sequence of SEQ ID NO: 14. In another embodiment, the recombinant vector does not comprise a Crt I enzyme gene, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20.

In a specific embodiment, the recombinant vector further comprises a Crt B enzyme gene and a Crt I enzyme gene. In a preferred embodiment, the Crt I enzyme gene encodes a Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 18. In another specific embodiment, the Crt I enzyme gene comprises a gene sequence of SEQ ID NO:9.

The present invention further provides a method for producing a Crt I-containing microorganism comprising: (a) constructing a recombinant vector comprising a genetically modified Crt I enzyme gene, wherein the genetically modified Crt I enzyme gene encodes the SEQ ID NO: 20 Crt I enzyme; (b) transgenic the recombinant vector into a microorganism; and (c) using a suitable culture method to cause the microorganism to express the genetically modified Crt I enzyme gene to produce a Crt I enzyme.

The present invention also provides a microorganism comprising a recombinant vector, wherein the recombinant vector comprises a Crt I enzyme gene, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20. Therefore, the microorganism can produce a genetically modified Crt I enzyme.

In one embodiment, the microorganism is a photosynthetic bacterium. In a preferred embodiment, the photosynthetic strain is Rhodovulum .

The present invention further provides a method for producing a Crt E-containing microorganism comprising: (a) constructing a recombinant vector comprising a genetically modified Crt E enzyme gene, wherein the genetically modified Crt E enzyme gene encodes the SEQ ID NO: 19 Crt E enzyme; (b) transgenic the recombinant vector into a microorganism; and (c) using a suitable culture method to cause the microorganism to express the genetically modified Crt E enzyme gene to produce Crt E enzyme. In a specific embodiment, the recombinant vector does not comprise a Crt I enzyme gene, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20.

The present invention also provides a microorganism comprising a recombinant vector, wherein the recombinant vector comprises a Crt E enzyme gene, wherein the Crt E enzyme gene encodes a Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 19. In a specific embodiment, the recombinant carrier does not include A Crt I enzyme gene is encoded, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20. Therefore, the microorganism can produce a genetically modified Crt E enzyme.

The present invention provides a method for producing a carotenoid comprising: (a) providing a recombinant vector containing an enzyme gene required for synthesizing a carotenoid, wherein the enzyme gene required for the synthesis of the carotenoid comprises a Crt E enzyme gene a Crt B enzyme gene and a Crt I enzyme gene, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20; (b) transducing the recombinant vector into a microorganism And (c) causing the microorganism to express an enzyme gene required for the synthetic carotenoid on the recombinant vector to produce a carotenoid.

In a specific embodiment, the Crt E enzyme gene encodes a Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 16 or 19. In a preferred embodiment, the Crt E enzyme gene encodes a Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 19. In another specific embodiment, the Crt E enzyme gene comprises a gene sequence of SEQ ID NO: 7 or 14. In a preferred embodiment, the Crt E enzyme gene comprises a gene sequence of SEQ ID NO: 14.

In another specific embodiment, the Crt B enzyme gene encodes a Crt B enzyme comprising an amino acid sequence of SEQ ID NO: 17. In a preferred embodiment, the Crt B enzyme gene comprises a gene sequence of SEQ ID NO: 8. The Crt B enzyme used in the present invention is a normal or wild type Crt B enzyme.

In one embodiment, the carotenoid is a water soluble carotenoid. In a preferred embodiment, the carotenoid is a lycopene. In a more preferred embodiment, the lycopene is a water soluble lycopene.

In one embodiment, the microorganism is a photosynthetic bacterium. In a preferred embodiment, the photosynthetic strain is Rhodovulum .

The present invention further provides a method for producing carotenoids, comprising: (a) providing a recombinant vector containing an enzyme gene required for synthesizing carotenoids, wherein the enzyme gene required for synthesizing the carotenoid comprises a Crt E enzyme a gene, a Crt B enzyme gene, and a Crt I enzyme gene, wherein the Crt E enzyme gene encodes a Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 19, wherein the Crt I enzyme gene does not encode a a Crt I enzyme of the amino acid sequence of SEQ ID NO: 20; (b) transposing the recombinant vector into a microorganism; and (c) causing the microorganism to exhibit the desired synthesis of the carotenoid on the recombinant vector The enzyme gene produces carotenoids. Thus, in the above method, only the Crt E enzyme gene is mutated to produce a modified Crt E enzyme, and the Crt I enzyme gene is a normal or wild type Crt I enzyme gene.

The present invention further provides a method for producing carotenoids, comprising: (a) providing a vector containing an enzyme gene required for synthesizing carotenoids, wherein the enzyme gene required for synthesizing the carotenoid comprises a Crt E enzyme gene, a Crt B enzyme gene and a Crt I enzyme gene, wherein the Crt E enzyme gene encodes a Crt E enzyme comprising SEQ ID NO: 16 or SEQ ID NO: 19, wherein the Crt I enzyme gene encodes SEQ ID NO: 18 Or the Crt I enzyme of SEQ ID NO: 20; (b) transducing the vector having the enzyme gene required for the synthesis of the carotenoid into a microorganism to produce a recombinant microorganism; and (c) using an appropriate culture method The microorganism that recombines the gene expresses the enzyme gene required for the synthesis of the carotenoid to produce a carotenoid.

Therefore, through the above method, the present invention at least Crt through genetic modification technology The E, Crt B and Crt I enzyme genes are transferred into a microorganism, wherein at least one of the Crt E and Crt I enzyme genes is a modified or mutated gene or both are modified or mutated genes, thereby improving Or a mutated Crt E and/or Crt I enzyme protein, that is, the present invention allows the microorganism to have at least one modified or mutated carotenoid enzyme protein in the body to promote the production of carotenoids by the microorganism, and The yield of carotene was significantly increased compared to the Crt E, Crt B and Crt I enzyme genes that were simply transfected into the wild type. In the present invention, the Crt E enzyme gene, the Crt B enzyme gene and the Crt I enzyme gene on the vector comprise a permutation combination of the following: (1) the Crt E enzyme gene encodes Crt E comprising SEQ ID NO: 19. The enzyme, and the Crt B enzyme gene and the Crt I enzyme gene encode the wild type Crt B and Crt I enzymes; (2) the Crt I enzyme gene encodes the Crt I enzyme containing SEQ ID NO: 20, and the Crt B enzyme gene. And the Crt E enzyme gene encodes a wild-type Crt B and Crt E enzyme; and (3) the Crt E enzyme gene encodes a Crt E enzyme comprising SEQ ID NO: 19, the Crt I enzyme gene encoding comprising SEQ ID NO The 20 Crt I enzyme, and the Crt B enzyme gene have a wild-type Crt B enzyme.

In a specific embodiment, when the Crt E enzyme gene encoding the Crt E enzyme of SEQ ID NO: 16 is encoded, the Crt I enzyme gene is not a gene encoding the Crt I enzyme comprising SEQ ID NO: 18. Therefore, the Crt E and Crt I enzyme genes are not wild-type Crt E and Crt I enzyme genes, i.e., wild-type Crt E and Crt I enzymes in the present invention.

In another embodiment, the enzyme gene required for the synthesis of the carotenoid further comprises a Crt Y, Crt Z, Crt G, Crt W and/or Crt X enzyme gene.

"Carotenoids" as used herein includes, but is not limited to, lycopene (lycopene), β-carotene, zeaxanthin, zeaxanthin β-D-diglucoside, caloxanthin, brow algae Calcium beta-D-diglucoside, nostoxanthin, astaxanthin, 2-hydroxy astaxanthin, 2, 2'-double 1,2'-dihydroxyastaxanthin, astaxanthin β-D-glucoside, astaxanthin β-D-diglucoside, 4,4'dione-β-carotene (canthaxanthin), 2-hydroxy-4,4'dione-β-carotene (2-hydroxy canthaxanthin), 2,2'-dihydroxy-4,4' Ketone-β-carotene (2,2'-dihydroxy canthaxanthin), β-apo-4'-caroteral, β-apo-8'-carrotaldehyde, β-apo-12'-carrotaldehyde, β -Apo-8'-carotenic acid, cryptoxanthin, citrine, lutein, volcanic acid, round yeast erythromycin-aldehyde, round yeast-erythroacetate, ethyl sulphate-ethyl ester or dehydrogenation Red truffle yellow, also includes natural carotene. In a preferred embodiment, the carotenoid is a lycopene.

In one embodiment, the carotenoid is a water soluble carotenoid. In another embodiment, the lycopene is a water soluble lycopene.

The term "transfer" as used herein includes, but is not limited to, the use of gene gun method, electroporation protoplast method, microneedle puncture method, liposome coating method and conjugated transfection (Conjugation) to transfer the target gene into The target organism is in the genome, thereby achieving the purpose of transforming the organism. In a preferred embodiment, the transformation is a conjugated transformation method.

The "microorganism" described herein may be a microorganism which itself produces carotenoids, that is, the microorganism itself has various enzymes and factors required for the production of carotenoids. And materials. Therefore, the present invention improves the two enzymes required for the synthesis of carotenoids, namely Crt E enzyme and Crt I enzyme, to increase the production of carotenoids produced by the microorganism.

The "microorganism" of the present invention is obtained from plants, animals, bacteria, fungi, insects, protozoa, viruses or mycoplasma. In addition, the term "microorganism" as used herein includes, but is not limited to, prokaryotic microorganisms and eukaryotic microbes. In one embodiment, the microorganism is a prokaryotic microorganism. In a preferred embodiment, the prokaryotic microorganism is a bacterium. In a more preferred embodiment, the bacterium comprises a Gram-positive bacterium and a Gram-negative bacterium.

In another embodiment, the bacterium is a Gram-negative bacterium. In a preferred embodiment, the Gram-negative strain is a photosynthetic bacterium.

In one embodiment, the microorganism is a photosynthetic bacterium. In a preferred embodiment, the photosynthetic strain is Rhodovulum .

The term "photosynthetic bacteria" as used herein includes, but is not limited to, bacteria that can utilize photosynthesis for light energy. In one specific embodiment, the photosynthetic bacteria include, but are not limited to Rhodospirillaceae (Rhodospirillaceae), thiophanate Red Branch (Chromatiaceae), tepidum Section (Chlorobiaceae) and sliding filamentous tepidum Section (Chloroflexaceae).

The present invention provides a method for preparing carotenoids, comprising the steps of: (a) cultivating a microorganism for producing carotenoids in a medium, wherein the microorganism secretes carotenoids in the medium, wherein the microorganisms have A microorganism for synthesizing a carotenoid enzyme gene, wherein the enzyme gene required for the synthesis of the carotenoid comprises a Crt E enzyme gene, a Crt B enzyme gene, and a Crt I enzyme gene, wherein the Crt E enzyme gene encodes a Crt E enzyme having SEQ ID NO: 16 or SEQ ID NO: 19, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising SEQ ID NO: 18 or SEQ ID NO: 20; (b) collecting the medium to Separating a carotenoid-containing culture solution; (c) adding a coagulant to the carotenoid-containing culture solution; and (d) treating the culture solution by solid-liquid separation, collecting and reacting with the aggregating agent to precipitate Solid carotenoids.

In one embodiment, the microorganism exhibits a Crt E enzyme comprising SEQ ID NO: 19 and/or a Crt I enzyme comprising SEQ ID NO: 20.

In one embodiment, the carotenoid is a lycopene. In a preferred embodiment, the carotenoid is a water soluble carotenoid.

In a specific embodiment, the aggregating agent includes, but is not limited to, a solution containing metal ions. In a preferred embodiment, the aggregating agent includes, but is not limited to, a solution of divalent metal ions. In a more preferred embodiment, the metal ion is a calcium metal ion.

In another embodiment, the aggregating agent includes, but is not limited to, an aqueous solution of magnesium sulfate, calcium chloride, zinc chloride or sodium chloride. In a preferred embodiment, the aggregating agent is an aqueous solution of calcium chloride or zinc chloride. In a more preferred embodiment, the aggregating agent is an aqueous solution of calcium monochloride.

In a specific embodiment, the medium includes, but is not limited to, a solid medium or a liquid medium. In a preferred embodiment, the medium is a liquid medium. In another preferred embodiment, the medium is a culture. Therefore, if the microorganism secreting water-soluble carotenoid is directly cultured using the culture solution, the method can be easily separated by centrifugation. The microorganism and the culture solution are separated, and the culture liquid containing the water-soluble carotenoid is directly obtained, and the carotenoid is obtained by agglutination and solid-liquid separation, which is advantageous for shortening and reducing the process and cost of the general carotenoid.

The method for carotenoid-containing microorganisms according to the present invention, wherein the carotenoids produced by the microorganisms include, but are not limited to, those used in the fields of medicine, health food, cosmetics, animal feed, fuel adjuvants and the like. In one embodiment, the carotenoid is used in the manufacture of cosmetics, skin care products, foods, health foods, health foods, animal drugs, or human drugs.

The method for preparing a carotenoid-containing microorganism provided by the invention has the following advantages when compared with other conventional techniques:

(1) The present invention utilizes a method of microbial transformation to transfer a modified carotenoid synthesis gene (such as Crt E or I) into a microorganism, which allows the microorganism to incrementally produce carotenoids (such as lycopene). The carotenoid content and purity in the microbial cell can be greatly improved.

(2) Compared with the method generally extracted from plants (such as tomato) or from other microorganisms, the present invention has the ability to obtain carotenoids (such as lycopene) from the microbial culture solution, which can achieve convenient extraction and yield. The advantage of high.

(3) The method of the present invention allows the microorganism to incrementally produce lycopene, and the lycopene can be converted into other carotenoids by other enzymes (as shown in Fig. 2), so that the present invention can make the upstream stevia red The increase in the yield of the prime, which means that the present invention is not only transferred to the microorganism In addition to the good lycopene synthesis gene (Crt E and/or Crt I), if another enzyme gene (Crt Y, Crt Z, Crt G, Crt W or Crt X) is further transferred into the microorganism, the upstream The production of lycopene is increased, so other enzyme genes only provide the function of converting the lycopene into other carotenoids, so the yield of the converted carotenoids will also increase.

Figure 1 is a schematic illustration of the lycopene synthesis pathway.

Figure 2 is a schematic representation of various carotenoid synthesis pathways.

Figure 3 is a design of a vector containing the Crt EBI gene. (A) is a pET23-a vector. (B) is the pBMTB-4 vector.

Figure 4 is a comparison of lycopene content in different mutants.

Figure 5 is a TLC analysis of the obtained lycopene.

Figure 6 shows the lycopene content of the engineered strain.

Figure 7 is a mass spectrometric identification of the obtained lycopene.

The spirit of the present invention is to enable microorganisms to function as carotenoids and to increase the production of carotenoids. In view of this, the invention includes, but is not limited to, the description above and below. The embodiment is shown in the following example.

Embodiment 1

Strain and culture conditions

The present invention is directed to a strain capable of producing carotenoids, and further a strain capable of producing lycopene, particularly a genetically modified photosynthetic bacterium. The present invention takes Rhodovulum as an example, and has the ability to produce lycopene, and the genetic modification method and basic culture conditions of photosynthetic bacteria are known techniques, please refer to literature such as FEMS Microbiol Lett (2006). 265: 69-75, U.S. Patent Application No. 10/585,419 and Hunter CN (J. Bacteriol (1994), 176(12): 3692-3697), and Taiwan Patent No.: I467018.

The original species of the genus Rhododendron can be taken from major biological resource centers or international strain preservation centers, such as strain ATCC 35886 (BCRC 16417), or self-screening photosynthetic bacteria. The obtained strain was cultured according to the standard procedure, and the following methods for genetic improvement were as follows: (1) The photosynthetic strain of the genus Rhododendron was cultured by selecting rifampicin (50 μg/ml), and the resistant colonies were screened and stored frozen; According to the implementation method of Taiwan Patent No.: I467018, the photosynthetic strain of C. elegans with the crtCD gene was obtained. (2) The common expression vector pET23a was used as the backbone, and the lycopene biosynthesis enzymes CrtE, CrtB and CrtI were sequentially A reverse transcription polymerase chain reaction (PCR) method is selected for the vector (the primers used in the present invention are SEQ ID NO: 1 to SEQ ID NO: 6; in addition, wild type (WT) CrtE, CrtB and The gene sequence of CrtI, which is SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 respectively, forms the construct pET23-a-CrtIBE, and then transfects the entire set of Crt IBE into the vector pBMTB- 4 of the EcoRI restriction enzyme cleavage site, which is labeled with tetracycline (as shown in Figure 3); and (3) modified erythromycin bacteria to produce lycopene: the above-mentioned Crt The vector constructed by IBE gene was conjugated to E. coli strain S17-1 for conjugation The strain was transformed into a photosynthetic strain of Xiaohongxue; and the strain was screened by rifampicin and tetracycline. Finally, the improved The pigment produced by the strain was identified as lycopene by chromatography, nuclear magnetic resonance, and mass spectrometry, and showed the ability to produce lycopene. The culture conditions of the genetically modified photosynthetic strain obtained by the above method are: growth temperature of 26 ° C, no need to illuminate; continuous aeration or shaking to provide sufficient oxygen.

Embodiment 2

Gene mutation

The lycopene biosynthesis gene mutation was performed by site-directed mutagenesis. The pET23-a vector containing the Crt IBE gene constructed above was used as a template, and the primers for the Crt E and Crt B genes were inserted into the forward and reverse directions of the mutation position (the primer sequence used for the Crt E mutation is SEQ ID NO). :10 and SEQ ID NO: 11, the primer sequence used for the Crt I mutation is SEQ ID NO: 12 and SEQ ID NO: 13), and then DNA polymerase is added and amplified by PCR. After 18 PCR cycles, the restriction enzyme DpnI was added at 37 ° C to decompose the original plastid.

After saturation-mutated linear plastids were ligase-ligated, they were first transformed into E. coli JM109 and smeared on LB plates, and the selection gene was Amp ^R . After overnight incubation in a 37 ° C incubator, the colonies were scraped off and the plastids were extracted. This is a plasmid library covering the Crt gene mutants. Next, the plastids of the plastid library were transformed into E. coli cells and smeared again on the LB plate. After overnight incubation in a 37 ° C incubator, the color change of the colonies was observed, and colonies with color changes were selected. The selected colonies were further cultured in LB medium, and cultured overnight in a 37 ° C incubator, and 4 ml of the bacterial solution was centrifuged, and then weighed, and the color change of the precipitate after centrifugation of each mutant was observed.

Embodiment 3

Comparison of lycopene production of Escherichia coli mutant

The E. coli mutant pellet was weighed and then washed with 200 μl of phosphate buffered saline (PBS). After PBS was removed by centrifugation, 50 μl of PBS was further added for reconstitution, followed by addition of 450 μl of n-hexane/acetone (1/1; v/v), and extraction at 25 ° C for 3 hours or more. Finally, it was centrifuged at 14,000 rpm for 5 minutes, and the upper layer (organic layer) was taken for analysis.

The thin layer chromatography analysis was carried out with a lycopene organic extract, wherein the mobile phase condition of the thin layer chromatography was n-hexane/acetone (10/1; v/v). 25 μl of the sample was spotted on the chromatographic sheet, and after running for several minutes in a closed glass cell, the generation and change of the layer band were observed, and the mutant of the high lycopene yield was selected for gene sequencing. After sequencing, the mutation site of the mutant was changed as follows: Crt E H234N (changing CAC to AAT, ie SEQ ID NO: 14) and Crt I I480V (changing ATT to GTG, ie SEQ ID NO: 15). In addition, the present invention also sequences the amino acid sequences of wild-type Crt E, Crt B and Crt I enzyme proteins, and the amino acid sequences of the modified Crt E and Crt I enzyme proteins, the sequences of which are as follows: (1) wild The amino acid sequence of the Crt E enzyme protein is SEQ ID NO: 16; (2) the amino acid sequence of the wild type Crt B enzyme protein is SEQ ID NO: 17; (3) the wild type Crt I enzyme protein The amino acid sequence is SEQ ID NO: 18; (4) the amino acid sequence of the modified Crt E enzyme protein is SEQ ID NO: 19; and (5) the amino acid sequence of the modified Crt I enzyme protein. Is SEQ ID NO: 20.

Using ImageJ software, the relative quantitative comparison of lycopene between the mutants was carried out by image contrast analysis based on the same amount of bacteria. As shown in Figure 4, single point mutants CrtE H234N and CrtI I480V compared to wild-type (wild-type Crt IBE genome) production. Both of them have more than 2 times the increase in lycopene production, while the double-point mutant (E&I double mutation; Crt E and I double mutation) has an additive effect, making the overall lycopene yield much higher than that of wild type. (WT) 6.9 times the yield.

Embodiment 4

Collecting photosynthetic bacteria culture solution and lycopene in the cells

The mutant plastids were conjugated to the genus Rhodobacter sphaeroides using E. coli strain S17-1 as a vector. The genetically modified photosynthetic bacteria are cultured in a culture solution, and the present invention finds that the culture solution gradually turns red, that is, the culture solution and the bacteria have lycopene, which means that the photosynthetic bacteria produce lycopene and some of the eggplant The erythromycin is secreted in the culture solution.

Another 1.8 ml of the culture solution was added, and 0.1 ml to 0.2 ml of an aqueous calcium chloride solution was added to be uniformly mixed; the calcium chloride culture solution was added from a clear clarification to a turbidity and a pigment agglutination. The agglutinated lycopene mixture can be obtained by centrifugation or filtration. For example, small centrifugation: 2 ml of the culture solution to which the agglutinating agent is added is placed in a centrifuge tube and centrifuged at 8000 g for 3 minutes in a table centrifuge, the pigment mixture. It is separated from the culture solution and precipitated on the bottom of the centrifuge tube. To further confirm the presence of lycopene, the lycopene organic extract was subjected to thin layer chromatography (TLC) analysis, in which the mobile phase condition of thin layer chromatography was n-hexane/acetone (10). /1;v/v). The pigment was analyzed by TLC to have the same Rf value as the lycopene standard.

In addition, the obtained cells containing the Crt IBE gene construct were extracted with acetone several times, and the extract was analyzed by TLC to find that the pigment type was very simple for subsequent purification, and the pigment was The Rf value is consistent with the lycopene standard (as shown in Figure 5).

In addition, the extract was analyzed for lycopene content by OD472, and the result of converting the lycopene to the dry weight ratio of the cells was as shown in Fig. 6. Figure 6 shows the original original host without any modification and it does not contain lycopene, but the construct containing the Crt IBE genome (ie wild type) and its mutation (CrtE mutant H234N and CrtI mutant I480V) The engineered constructs (ie, Crt E mutation and Crt I mutation) were sent to the photosynthetic strain to obtain an excellent lycopene content, which accounted for a dry weight ratio (%) of 1.65, and Crt E and Crt The single point mutation of I does increase the production of lycopene over the genome of the wild strain. From the above results, it can be reasonably predicted that the two-point mutation of Crt E and Crt I will further increase the production of lycopene.

Embodiment 5

Lycopene extraction, purification and preservation

The obtained lycopene crude product was dissolved in a n-hexane/acetone mixture, and the mixture was poured into a 1 liter extraction flask and allowed to stand at -20 ° C for stratification. After the layers were separated, the upper layer of the mixture was separated and water was removed with anhydrous magnesium sulfate. After filtration through a filter paper, the filtrate was drained with a reduced pressure rotary concentrator. The dried solid was re-dissolved in a small amount of diethyl ether (10-20 mL).

Embodiment 6

Lycopene chemical composition analysis

The isolated lycopene product was identified by nuclear magnetic resonance and mass spectrometry using a nuclear magnetic resonance apparatus (BRUKER AVANCE-500 NMR SPECTROMETER, Germany) and Spectrometer (Waters Quattro micro API MS) identification. The results of chemical identification of lycopene of the present invention can be confirmed by literature comparison (Advanced Synthesis and Catalysis (2008), 350: 365-369).

As shown in Figure 7, the chemical identification data of lycopene is as follows: ¹ H NMR: δ 5.9-6.7 (m, 16 H, double bond H), δ 5.11 (m, 2 H, double bond H), δ 2.11 ( m, 8 H, 4 X CH ₂ ), δ 1.97 (s, 12 H, 4 X CH ₃ ), δ 1.82 (s, 6 H, 2 X CH ₃ ), δ 1.68 (s, 6 H, 2 X CH ₃ ), δ 1.61 (s, 6 H, 2 X CH ₃ ); HR-MS (EI): m/z = 536.3, calculated as C ₄₀ H ₅₆ .

The detailed description of the preferred embodiments of the present invention is not intended to limit the scope of the present invention, and it is intended to be The modifications are intended to be included in the scope of the patent application of the present invention.

<110> Honglintang Biotechnology Co., Ltd.

<120> Microorganisms for producing carotenoids and their applications

<130> 2470-BNS-TW

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<170> PatentIn version 3.5

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Claims (10)

A Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20. A recombinant vector comprising a Crt I enzyme gene, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20. The recombinant vector of claim 2, further comprising a Crt E enzyme gene and a Crt B enzyme gene. The recombinant vector of claim 3, wherein the Crt E enzyme gene encodes a Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 19. A microorganism comprising the recombinant vector of claim 2 of the patent application. A method for producing carotenoids, comprising: (a) providing a recombinant vector containing an enzyme gene required for synthesizing carotenoids, wherein the enzyme gene required for synthesizing the carotenoid comprises a Crt E enzyme gene, a Crt a B enzyme gene and a Crt I enzyme gene, wherein the Crt I enzyme gene encodes a Crt I enzyme comprising an amino acid sequence of SEQ ID NO: 20; (b) transducing the recombinant vector into a microorganism; (c) causing the microorganism to express the enzyme gene required for the synthetic carotenoid on the recombinant vector to produce a carotenoid. The method of claim 6, wherein the Crt E enzyme gene encodes a Crt E enzyme comprising an amino acid sequence of SEQ ID NO: 19. The method of claim 6, wherein the carotenoid is a lycopene. The method of claim 6, wherein the microorganism is a photosynthetic bacteria. The method of claim 9, wherein the photosynthetic strain is Rhodovulum .