# Patent

## Title
Method of producing lycopene using recombinant Escherichia coli

## Abstract

A method of producing lycopene with high productivity by means of a recombinant bacterial strain includes preparing the recombinant vector containing genes encoding proteins, which are required for lycopene biosynthesis. The genes involved in lycopene biosynthesis are crtE, crtB and crtI, and at least one of the said three genes (crtE, crtB and crtI) is selected from the group consisting of crtE with the nucleotide Sequence 1, crtB with the nucleotide Sequence 3 and crtI with the nucleotide Sequence 5, of the Sequence List. The said recombinant vector is transformed into Escherichia coli.

## Background
The present disclosure is the United States National Stage Entry of International Application No. PCT/KR2008/002535 having a filing date of May 6, 2008, which claims priority to Korean Patent Application No. 10-2007-0043682 having a filing date of May 4, 2007, which is incorporated herein in its entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 16, 2010, is named CNS3PCUS.txt, and is 18,161 bytes in size.
TECHNICAL FIELD
The present invention relates to the method of producing highly concentrated lycopene by means of a bacterial strain that is transformed with a vector containing the genes encoding the proteins involved in lycopene biosynthesis. In more detail, the present invention relates to the method of producing lycopene with enhanced productivity, by culturing and fermenting transformed E. coli, under a specified condition, which has an introduced vector containing the genes required for lycopene biosynthesis, wherein the vector is prepared by combining at least one of the following new genes: crtE with the nucleotide Sequence 1, crtB with the nucleotide Sequence 3 and crtI with the nucleotide Sequence 5, of the Sequence List.
BACKGROUND ART
Lycopene has the structure shown in Chemical Structure 1, and can be obtained with a yield of 0.02 g from 1 kg of tomato. Lycopene, which is responsible for the red color of tomato, watermelon, grape, etc, is a lipid-soluble substance with very low polarity, and has strong antioxidant and anticancer effects.




Hereunder is the summary of the previous studies about the art. In 2000, it was reported by Omer's group at the Carmanos Cancer institute in Detroit that lycopene suppressed the metastasis of prostate cancer (Omer Kucuk et al., Cancer Epidemiology, 10, 861-869, 2001). The effect of lycopene in releasing the symptom of patients with exercises-induced asthma was proved by the LycoRed, which is a specialized maker of lycopene, and by an allergy lab at the Hasharon hospital in Tel Aviv (I. Neuman et al., Allergy, 55, 1184-1189). Lycopene was also shown to have an excellent protective effect against myocardial disease and atherosclerosis, according to the clinical experiments carried out by the Research Institute of Public Health of the University of Cuopio in Finland. (Tiina Rissanen et al., Exp Biol Med (Maywood), 227, 900-907, 2002).
As the excellent effects of carotenoids were proved as said, there has been increasing needs for lycopene. Lycopene has been produced by extraction directly from natural sources or by organic synthesis, while recently there have been researches actively going on to produce it by means of microorganism. Specifically there are two types of method which involve the cultivation and fermentation of microorganism to produce lycopene: (1) the method comprising the introduction of the genes which are required for lycopene biosynthesis into a bacterial strain which does not produce lycopene, (2) the method comprising the inactivation of lycopene cyclase of the bacterial strains which produce lycopene as an intermediate metabolic product of the biosynthesis of carotenoids such as carotene or astaxanthin.
The process of lycopene biosynthesis is illustrated in FIG. 1.
Glucose or glycerol are converted into isopentenyl pyrophosphate (hereafter IPP) or dimethylallyl pyrophosphate (hereafter DMAPP), via metabolic pathways such as 2-C-methyl-D-erythritol-4-phosphate pathway (hereafter MEP pathway) or the mevalonate pathway (hereafter MVA pathway). The present invention adopted MVA pathway for the biosynthesis of IPP. Via MVA pathway, glyceraldehyde-3-phosphate derived from glucose or glycerol is converted into Acetyl-CoA, which then undergoes a series of conversions into acetoacetyl-CoA, 3-hydroxy-3-methylglutaryl Coenzyme A (hereafter HMG-CoA), mevalonate, mevalonate-5-phosphate, mevalonate-5-diphosphate and eventually into IPP. The genes which encode the enzymes required for this process are atoB, mvaS, mvaA, mvaK1, mvaK2 and mvaD. The IPP thus synthesized undergoes a series of conversions to become farnesyl pyrophosphate (hereafter FPP), which is an important intermediate metabolic product of isoprenoid pathway. FPP is then converted into geranylgeranyl pyrophosphate (hereafter GGPP), which then into phytoene, which then finally converted into lycopene. The genes encoding the enzymes involved in this process are crtE, crtB and crtI.
As said, the mevalonate pathway and the non-mevalonate pathway are known as bio- synthetic pathways of IPP, which is a common precursor of carotenoids, and the mevalonate pathway is known to exist in most eukaryotes (e.g., Saccharomyces cerevisiae), cytoplasm of plant cells, some bacteria (e.g., Streptococcus pneumoniae and Paracoccus zeaxanthinifaciens) and malarial cell. The non-mevalonate pathway exists in most bacteria (e.g., E. coil) and plastid of plant cells. Thus E. coli, which is a Gram-negative bacterium, biosynthesizes IPP only via the non-mevalonate pathway. Wild-type E. coil, however, has no genes required for the biosynthesis of carotenoids such as lycopene, and so can not produce lycopene.
As to the production of lycopene from the strains which does not produce lycopene, most studies have concerned either the finding of new genes involved in lycopene biosynthesis or the recombination of previously known genes, with a focus on the use of these genes for the biosynthesis of lycopene in E. coli or Saccharomyces cerevisiae. Roche Vitamins, Inc has made a strain of E. coli which has the lycopene content of 0.5 mg/gDCW by introducing crtB, crtI and crtE, which are the genes from Fla-vobacterium sp. R1534 (Luis Pasamontes and Yuri Tsygankov, US20040058410, 2004). Amoco Corporation has made a strain of Saccharomyces cerevisiae which has the lycopene content of 0.1 mg/gDCW, by using the crtI gene from Erwinia herbicola (Ausich, Rodney L. et al., U.S. Pat. No. 5,530,189, 1996). And Kirin Beer Kabushiki Kaisha has established a strain of E. coli which has the lycopene content of 2.0 mg/gDCW, by using the crtE, crtI and crtB genes from Erwinia uredovora (Narihiko Misawa et al., U.S. Pat. No. 5,429,939, 1995). Also a strain of transformed Candida utilis IFO 0988 with the lycopene content of 2.9 mg/gDCW was successfully prepared and cultured; which has the crtE, crtB and crtI genes from Erwinia uredovora, as well as the gene encoding HMG-CoA reductase of Candida utilis (Narihiko Misawa et al., J. of Biotechnology, 59, 169-181, 1998). Recently, a strain of recombinant E. coil obtained by transformation with crtE (encoding GGPP synthase), crtB (encoding phytoene synthase) and crtI (encoding phytoene desaturase), which are the carotenoid genes cloned from bacterial strains of Agrobacterium aurantiacum, Erwinia herbicola and Erwinia uredovora, has been reported to have the ability to biosynthesize lycopene (U.S. Pat. No. 6,706,516; Misawa and Shimada, J.Biotechnol., 59:169-181, 1998).
The yields of the studies, however, are very low, hindering the development of economical processes for lycopene production. To resolve this problem, the present invention employs new genes as well as combination of genes, providing the method of lycopene production with improved productivity using a transformed microorganism, wherein E. coli is transformed with atoB, mvaS, mvaA, mvaK1, mvaK2 and mvaD, which are the genes encoding the enzymes involved in the mevalonate pathway.
Thus the present invention is the result of the efforts to improve the lycopene productivity: For this, the crtE, crtB and crtI, which are some of the genes involved in lycopene biosynthesis, were isolated from sea metagenome and cloned, and their nucleotide sequences were determined; then the genes were introduced into vector in order to produce lycopene in a microorganism which does not produce lycopene, while the lycopen productivity was improved by combining new genes with previously known genes. In addition, lycopene productivity was further enhanced by introducing into E. coli the genes involved in the mevalonate pathway, thereby enabling E. coli to use the mevalonate pathway. Also a fermentation method was developed and provided to produce highly concentrated lycopene in the recombinant microorganism under a specified condition; the higher productivity of the strain than that of previous studies has been confirmed, and the present invention was accomplished thereupon.
DISCLOSURE OF INVENTION
Technical Problem
Accordingly, the present invention is directed to a method of producing lycopene efficiently from recombinant bacterial strain which is constructed using the recombinant vector containing new genes encoding the proteins required for lycopene biosynthesis.
According to an aspect of the present invention, the method of producing lycopene includes steps of preparing a recombinant vector containing genes encoding proteins, which are required for lycopene biosynthesis, wherein the genes involved in lycopen biosynthesis are crtE, crtB and crtI and at least one of the three genes (crtE, crtB and crtI) is selected from the group consisting of crtE with the nucleotide Sequence 1, crtB with the nucleotide Sequence 3 and crtI with the nucleotide Sequence 5, of the Sequence List; transforming the recombinant vector into E. coli; and culturing the E. coli transformant and recovering of lycopene from the culture medium.
Hereunder, the present invention will be described in more detail.
The present invention involves the cloning of crtE, crtB and crtI from sea metagenome, which are the 3 new genes encoding the proteins required for lycopene biosynthesis; the establishment of recombinant vector containing these genes; and the transformation with the recombinant vector of E. coli that does not produce lycopene.
Furthermore, the possibility of producing highly concentrated lycopene is proved by fermenting the E. coli which is transformed with the recombinant vector containing at least one of the new genes; preferably a combination of the new crtB (Sequence 3) and crtI (Sequence 5) genes with previously known crtE gene is proved to give higher productivity than that of previous studies; and thereupon the present invention has been accomplished.
The new genes of the present invention, each of which encoding a protein required for lycopene biosynthesis with a nucleotide sequence of either Sequence 1, Sequence 3 or Sequence 5, are obtained from sea metagenome. The Sequence 1, Sequence 2 and Sequence 3 encode following amino acid sequences, respectively: Sequence 2 (GGPP synthase), Sequence 4 (phytoene synthase) and Sequence 6 (phytoene desaturase).
The genes provided by the present invention may be transformed into a variety of host cells to be used usefully for lycopene production. Each of the new genes may be used separately, or more than 2 of them used together. For example, crtI of the present invention may be transformed into a microorganism which has only crtE and crtB to produce lycopene; also crtE, crtB and crtI of the present invention may be transformed into a microorganism which biosynthesizes carotenoids such as astaxanthin to attain better yield.
The present invention provides the method of producing lycopene in E. coli via the mevalonate pathway, by using lycopene biosynthesis genes along with the genes involved in the mevalonate pathway.
The method of the present invention comprising producing lycopene in microorganism using lycopene biosynthesis genes involves following 3 steps: (1) preparation of the recombinant vector which contains mvaK1, mvaD, mvaK2, mvaE, mvaS and idi in addition to the genes encoding the proteins required for lycopene biosynthesis, wherein the genes involved in lycopene biosynthesis are crtE, crtB and crtI and at least one of the three genes (crtE, crtB and crtI) is selected from the following group of genes: crtE with the nucleotide Sequence 1, crtB with the nucleotide Sequence 3 and crtI with the nucleotide Sequence 5, of the Sequence List; (2) trans-formation of the recombinant vector into E. coli; (3) culture of the E. coli transformant and recovering of lycopene from the culture medium.
The mvaK, mvaD, mvaK2, mvaE, mvaS, crtE, crtB, crtI and idi genes, which are required for the biosynthesis of lycopene, may be used for the construction of at least one or more than one separate vectors, wherein each vector contains at least one gene selected from the same genes; For example, each of mvaK, mvaD, mvaK2, mvaE, mvaS, crtE, crtB, crtI and idi genes may be either separately used to construct 9 vectors or to construct several vectors which contain at least one of the genes, to be used for transformation. The combination of the vectors, which are constructed separately, however, should be in such a way that the transformed E. coli has all the mvaK, mvaD, mvaK2, mvaE, mvaS, crtE, crtB, crtI and idi genes. The mvaE of the present disclosure is a gene which has functions of both atoB and mvaA in the process of lycopene biosynthesis shown in FIG. 2.
Preferably, vectors may be constructed separately so that one vector has mvaK, mvaD, mvaK2, mvaE and mvaS genes and the other has crtE, crtB, crtI and idi, to be transformed into E. coli; wherein, the mvaK, mvaD, mvaK2, mvaE and mvaS genes may be from previously known genes, while at least one of the crtE, crtB and crtI genes may be from sea metagenome.
As a preferred practice of the present invention, the recombinant vector containing crtE, crtB, crtI and idi genes has following genes: the crtE gene with Sequence 7, the crtB gene with Sequence 3, the crtI gene with Sequence 5 and the idi gene of E. coli. As another preferred practice of the present invention, the recombinant vector containing crtE, crtB, crtI and idi genes has following genes: the crtE gene with Sequence 8, the crtB gene with Sequence 3, the crtI gene with Sequence 5 and the idi gene of E. coli. As still another preferred practice of the present invention, the recombinant vector containing crtE, crtB, crtI and idi genes has following genes: the crtE gene from Erwinia herbicola, the crtB gene with Sequence 3, the crtI gene with Sequence 5 and the idi gene of E. coli. 
The present invention is not limited to the combination of genes through the construction of certain types of recombinant vectors, but also includes any method of constructing a strain of E. coli that has mvaK, mvaD, mvaK2, mvaE, mvaS, crtE, crtB, crtI and idi genes, wherein at least one of crtE, crtB and crtI is the gene of the present invention obtained from sea metagenome.
In more detail, to produce highly concentrated lycopene from the culture of the reccmbinant strain, the basic culture conditions are optimized as follows: culture temperature is 25-35° C., pH of culture medium before autoclaving 7.0-7.5, shaking speed between 200 rpm and 1,000 rpm, and the concentration of dissolved oxygen between 20% and 30%.
On the other hand, the fed-batch culture is desirable to improve the growth of cells and the yield of lycopene. Culture medium is supplied whenever carbon sources are depleted; wherein the supply is controlled so that 0.4% of culture medium is added for each supply, and the composition of the culture medium is 70% of glycerol and 2% of MgSO4.H2O.
The amount of lycopene produced by the strain which is transformed according to the methods is measured for the present invention: That of the transformed E. coli which has the combination of the new crtE, crtB and crtI genes provided by the invention is 0.65 mg/L/hr; while that of the transformed E. coli which has the combination of at least one of the new genes of the invention with previously known genes is up to 3.30 mg/L/hr, which is increased to 9.7 mg/L/hr if fed-batch culture is employed, and to 36.5 mg/L/hr if additional IPTG induction is carried out.
In order to accomplish the object of the present invention, as described in detail: the mevalonate pathway was introduced; the optimum combination of genes was determined; fermentation condition was optimized through the fermentation of the recombinant E. coli prepared according to the method; and the higher productivity than that of previous inventions is established, providing an advantage over the previous inventions. The previous inventions give high yield but require much time, while the present invention gives high yield in a short time, making possible the development of economical process.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a process of lycopene biosynthesis.
FIG. 2 is the structure of recombinant vector pSANF.
FIG. 3 is the structure of recombinant vector pT5-ErEBI.
FIG. 4 is the structure of recombinant vector pT5-LYC-idi.
FIG. 5 is the structure of recombinant vector pT5-ErBI.
FIG. 6 is the structure of recombinant vector pT-EF5.
FIG. 7 is the structure of recombinant vector pT-RF5.
FIG. 8 is the structure of recombinant vector pT-SF5.



BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is described in more detail with respect to examples hereunder. The examples hereunder, however, are only for the purpose of illustrating the present invention, and the scope of the present invention is not limited to the examples hereunder.
EXAMPLE 1
Cloning of the Genes Involved in the Lycopene Biosynthesis and the Preparation of the Recombinant Vector pSANF
Wild-type E. coli does not produce lycopene, so mvaK1, mvaD, mvaK2, mvaE, mvaS, crtE, crtB, crtI and idi genes, which are involved in the production of mevalonate, IPP and lycopene, were introduced for lycopene production. Initially these genes were introduced into 2 vectors as follows: mvaK1, mvaD, mvaK2, mvaE and mvaS genes were introduced into pSTV28, while crtE, crtB, crtI and idi genes into pTrc99A. pSTV28 has the chloramphenicol resistance and pTrc99A the ampicillin resistance, so the recombinant strain containing both vectors together could be selected using both antibiotics.
Of those genes, mvaK1, mvaD, mvaK2, mvaE, mvaS and idi genes were from the genes whose sequences are already known, while crtB and crtI genes were selected from sea metagenome. The crtE gene from metagenome and that from previously known genes were separately used in order to measure lycopene productivity.
The mvaK1 gene was cloned from Staphylococcus aureus ATCC35556. The DNA fragment amplified from the genome DNA of Staphylococcus aureus ATCC35556 was digested with EcoRI and Sad, followed by insertion into pSTV28 vector that was digested at the same restriction sites, and the resulting recombinant vector was named pSTV28-SA1.
The mvaD and mvaK2 genes were cloned from the genome DNA of Streptococcus pneumoniae. The mvaD gene was digested with Sad and KpnI restriction enzymes, followed by insertion into pSTV28-SA1 vector that was digested at the same restriction sites, and the resulting recombinant vector was named pSTV28-SA1D. The mvaK2 gene was digested with KpnI and BamH, followed by insertion into pSTV28-SA1D vector that was digested at the same restriction sites, and the resulting recombinant vector was named pSTV28-SA12D.
Before the insertion of mvaE and mvaS, the existence of promoter and transcription termination sequence were checked to carry out the over lap extension PCR, in order to join the DNA fragment containing the promoter with the DNA fragment containing the transcription termination sequence into a single fragment. The DNA fragment thus obtained was digested with BglII and NsiI, followed by insertion into pSTV28-SA12D vector that was digested with BamHI and PstI, and the resulting recombinant vector was named pSTV28-SA12D-Trc.
The mvaE and mvaS genes was cloned from the genome DNA of Enterococcus faecalis ATCC700802, and then subjected to the over lap extension PCR mvaE is a gene which functions as both atoB and mvaA in the process of lycopene biosynthesis. The DNA fragment containing mvaE and mvaS, which was obtained as said, was digested with BamHI and PstI, followed by insertion into pSTV28-SA12D-Trc vector that was digested at the same restriction sites, and the resulting recombinant vector was named pSANF <FIG. 2>.
The 5 genes were all purified with Qiagen PCR purification kit (Qiagen) before being used.
EXAMPLE 2
Cloning of the Genes Involved in the Lycopene Biosynthesis and the Construction of the Recombinant Vector pT5-LYC-idi
2.1 Cloning of crtE, crtB and crtI, which are the New Genes Involved in Lycopene Biosynthesis, from Sea Megagenome.
In order to obtain crtE, crtB and crtI, which are the genes involved in lycopene biosynthesis, genome DNA (metagenome) was obtained directly from the sea water and used to establish a library; and the clones with red color was selected taking an advantage of the red color of lycopene, followed by the DNA analysis of the clones.
To obtain metagenome DNA from sea, concentrated microorganisms were first obtained by concentrating large amount of sea water through membrane filtration. Since most microorganisms have the sizes between 0.2 μm and 10 μm, the large amount of sea water was initially passed through a filter with a pore size of 10 μm to remove various floating substances whose sizes are larger than 10 μm, and then the microorganisms whose sizes are lager than 0.2 μm are selectively collected using a filter with a pore size of 0.2 μm. Chromosomal DNA was isolated from the collected microorganisms by a method that uses CTAB (Hexadecyltrimethyl ammonium bromide) (Zhou et al., Appl. Environm. Microbiol. 62:316-322, 1996).
A library was constructed from the metagenome DNA which was obtained from the microorganisms, using a Copy Control fosmid library production kit (Epicenter) according to the manufacturer's manual. The kit used for library construction was Fosmid Vector Copy Control pCC1FOS (Epicenter). Ligation was carried out between the DNA to be inserted and the Copy Control vector pCC1FOS, and the ligated fosmid clones were subjected to packaging using the MaxPlax lambda packaging extracts (Epicenter). More than 10,000 clones could be obtained through the procedure.
The fosmid clones obtained as said was subjected to the stationary culture for 48 hours, then the color of the colonies was observed and red colonies selected. In order to check the existence of crt gene by PCR, primers were synthesized based on the conserved amino acid sequences in the C-terminus (crtIf) and the middle region of crtB (crtBr), of Erwinia uredovora, Erwinia herbicola, Flavobacterium sp. Strain ATCC21588, Rhodobacter sphaeroides and Agrobacterium aurantiacum. The nucleotide sequences of these primers are as follows:










crtIf:
5′-GTNGGNGCRGGCACNCAYCC-3′



 



crtBr:
5′-TCGCGRGCRATRTTSGTSARRTG-3′






The crt gene was amplified using the primers synthesized as said, while the template fosmid DNA was isolated from each of the red colonies. In detail, 100 ng of the fosmid DNA template was denatured at 94° C. for 5 minutes, followed by 20 cycles of ‘at 94° C. for 30 seconds->at 50° C. ̃60° C. for 30 seconds->at 72° C. for 1 minute’, then followed by 15 cycles of ‘at 94° C. for 30 seconds->at 50° C. for 30 seconds->at 72° C. for 1 minute’. As a result, an expected band with a size of 620 by could be obtained from one colony, which was inserted into pST-Blue1 vector (Nwagen), and then subjected to nucleotide sequence analysis and was found to have a homology with the previously reported nucleotide sequence of crtB gene.
A cluster of the whole lycopene biosynthesis genes that includes the crtB gene was attempted to be obtained by means of southern blot, using the fragment of crtB gene obtained as said as a probe. The fragment of crtB used as a probe had a DIG compound attached by PCR, while the template DNA was digested with BamHI, SalI and EcoRI restriction enzymes before southern blot. The DNA treated with each of various restriction enzymes was first subjected to electrophoresis on 0.97% agarose gel to separate the fragments according to their sizes, which were then transferred to nitrocellulose membrane (Schelicher & Schuell, Germany) by capillary transfer method. Hybridization was performed at 42° C. for more than 6 hours in a standard solution containing 50% formamide (5×SSC, 0.1% N-Lauroylsarcosine, 0.02% SDS, 5% Blocking regent, 50% Formamide) with the probe added. The membrane was subjected to binding with an antibody against the DIG to which alkaline phosphotase is attached; then the substrates NBT and X-phosphate were added to induce color development according to the manual of the manufacturer (Boehringer-Mannheim, Germany).
A band with a size of about 4 kb among the EcoR1 fragment DNA which gave a signal upon southern blot was inserted into the pBluescript II KS(+) vector (Stratagene) for DNA sequence analysis. The sequence analysis proved that the band has a region of about 3.2 kb in total containing crtE, crtB and crtI.
As said, crtE, crtB and crtI genes were cloned from sea metagenome and have different nucleotide sequences from those of previously known genes. The idi gene encoding IPP isomerase was amplified from the genome DNA of E. coli MG1655.
2.2 Construction of Recombinant Vector pT5-LYC-idi
The DNA fragment containing the crtE gene obtained as said was first digested with EcoRI and BamHI, followed by insertion into the pTrc99A vector digested at the same restriction sites, and the resulting recombinant vector was named pT-f5crtE. A DNA fragment of about 2.4 kb which contains whole the crtB and crtI genes was obtained through the over lap extension PCR reaction, which was purified using the Qiagen PCR purification kit and then digested with XhoI and SalI, followed by insertion into pT-f5crtE digested at the same restriction sites, and the resulting recombinant vector was named pT-f5EBI. The idi gene from E. coli MG1655 was digested with Sad and NotI, followed by insertion into pT-f5EBI digested at the same restriction sites, and the resulting recombinant vector was named pT5-LYC-idi (FIG. 4).
All the genes were purified using the Qiagen PCR purification kit (Qiagen) before being used.
EXAMPLE 3
Evaluation of Lycopene Productivity of E. coli Transformed with the Vector which Contains a Combination of the crtE, crtB and crtI Genes from Sea Megagenome with the Mevalonate Synthesis Genes
Lycopene biosynthesis was tested with the E. coli transformed with the pSANF and pT5-LYC-idi vectors of Example 1 and Example 2.
Initially MG1655 was transformed with the pSANF and pT5-LYC-idi vectors. A single colony of the transformed E. coli was obtained and inoculated into 5 mL of LB medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl) which contains 50 ppm of ampicillin and 20 ppm of chloramphenicol, followed by the shaking culture at 37° C. for 12 hours; then a 600 ml aliquot of the resulting culture medium was inoculated into 30 mL of 2YT medium which contains 1% of glycerol, 50 ppm of ampicillin and 20 ppm of chloramphenicol, followed by the main culture at 30° C. for 48 hours.
Upon the completion of the culture, an appropriate amount of the culture medium was taken to check lycopene productivity; by calculating dry cell weight (gDCW/L), yield (mg Lycopene/L, hereafter mg/L), content (mg Lycopene/gDCW, hereafter mg/gDCW) and productivity (mg Lycopene/L/hr, hereafter mg/L/hr).
In order to measure dry cell weight, 5 mL of the culture medium was taken and subjected to centrifugation in a 50 mL tube at 8,000 rpm for 10 minutes, and the cells were recovered by removing the supernatant. The recovered cells were suspended in 20 mL of sterilized distilled water and centrifuged to recover the cells that were cleansed of the components of the medium; then the recovered cells were completely suspended in 5 mL of sterilized distilled water and transferred to an aluminum weighing dish whose weight had been already measured. At this time, the centrifuge tube was washed with sterilized distilled water and the wash was added also to the weighing dish. The weighing dish was dried at 105° C. for more than 12 hours in a dry oven, followed by cooling, and then weighed to an accuracy of mg. Dry cell weight (gDCW/L) was calculated using Formula 1 below.

Dry Cell Weight(gDCW/L)={Weight of Dish with Dried Cells(mg)Weight of Dish(mg)}/5  [Formula 1]

The yield of lycopene was measured as follows: In order to extract lycopene, cells obtained from 100 μl of the culture medium by centrifugation was suspended in 400 μl of acetone, which was incubated at 55° C. for 15 minutes, which then 600 μl of acetone was added to and again incubated at 55° C. for 15 minutes. The extract was centrifuged at 14,000 rpm for 10 minutes, then the removed supernatant was measured of absorbance at the wavelength of 474.5 nm using a spectrophotometer. The measured value was substituted into an equation, which was obtained based on standard curve, to calculate the amount of lycopene based on the dilution rate. In order to obtain the standard curve, the standard lycopene purchased from Sigma was dissolved in acetone, the resulting solution was diluted to different concentrations of lycopene into acetone, and then the absorbances of the dilutes measured using a spectrophotometer at the wavelength of 474.5 nm was used to make the standard curve.
Lycopene Content (mg/gDCW) was calculated using Formula 2 based on the Dry Cell Weight and the Yield of lycopene.

Lycopene Content(mg/gDCW)=Yield of lycopene(mg/L)/Dry Cell Weight(gDCW/L)  [Formula 2]

Lycopene Productivity (mg/L/hr) was calculated using Formula 3, by dividing the Yield of lycopene, which was obtained as said, by incubation time.

Productivity(mg/L/hr)=Yield of lycopene(mg/L)/Incubation time(hr)  [Formula 3]

The Lycopene Productivity of the E. coli transformed with pSANF and pT5-LYC-idi, as determined according the method, is shown in Table 1.
Table 1












TABLE 1







Dry Cell Weight
Yield
Content
Productivity



(gDCW/L)
(mg/L)
(mg/gDCW)
(mg/L/hr)









5.11
31.2
6.1
0.65










EXAMPLE 4
Evaluation of Lycopene Productivity of E. coli Transformed with the Vector which Contains Either the crtE, crtB and crtI Genes from Erwinia herbicola or the Combination of the Genes with the Mevalonate Synthesis Genes
4.1 Evaluation of Lycopene Productivity of E. coli Transformed with the Vector which Contains the crtE, crtB and crtI Genes from Erwinia herbicola. 
The crtE, crtB and crtI genes from Erwinia herbicola were obtained to construct pT5-ErEBI (FIG. 3), which was transformed into E. coli to evaluate lycopene productivity by the same method of Example 3. The lycopene productivity of the transformed E. coli measured after 48 hours of culture was shown in Table 2.
Table 2












TABLE 2







Dry Cell Weight
Yield
Content
Productivity



(gDCW/L)
(mg/L)
(mg/gDCW)
(mg/L/hr)









3.7
12.7
3.5
0.3










4.2 Evaluation of Lycopene Productivity of E. coli Transformed with the Vector which Contains the Combination of the crtE, crtB and crtI Genes from Erwinia herbicola with the Mevalonate Synthesis Genes.
The pT5-ErEBI containing the crtE, crtB and crtI genes from Erwinia herbicola and the pSANF of Example 1 were transformed into E. coli to evaluate lycopene productivity by the same method of Example 3. The lycopene productivity of the transformed E. coli measured after 48 hours of culture was shown in Table 3.
Table 3












TABLE 3







Dry Cell Weight
Yield
Content
Productivity



(gDCW/L)
(mg/L)
(mg/gDCW)
(mg/L/hr)









4.0
66.3
16.6
1.4










EXAMPLE 5
Evaluation of Lycopene Productivity of E. coli Transformed with the Vector which Contains the Combination of the crtE Gene from Sea Metagenome and the crtB and crtI Genes from Erwinia herbicola with the Mevalonate Synthesis Genes
The crtB and crtI genes in the vector pT5-LYC-idi of the Example 2 were substituted by the already known same genes from Erwinia herbicola to construct recombinant vector pT5-ErBI (FIG. 5), which was transformed into E. coli to evaluate lycopene productivity by the same method of Example 3.
The lycopene productivity of the E. coli transformed with pSANF and pT5-ErBI measured after 48 hours of culture was shown in Table 4.
Table 4












TABLE 4







Dry Cell Weight
Yield
Content
Productivity



(gDCW/L)
(mg/L)
(mg/gDCW)
(mg/L/hr)









5.89
50.1
8.5
1.04










EXAMPLE 6
Evaluation of Lycopene Productivity of E. coli Transformed with the Vector which Contains the Combination of the crtE Gene from Erwinia herbicola (AMOCO CORPORATION, U.S. Pat. No. 5,530,189) and the crtB and crtI Genes from Sea Metagenome with the Mevalonate Synthesis Genes
The crtE gene in the vector pT5-LYC-idi of the Example 2 was substituted by a same gene from Erwinia herbicola to construct recombinant vector pT-EF5 (FIG. 6), which was transformed into E. coli to evaluate lycopene productivity by the same method of Example 3.
The lycopene productivity of the E. coli transformed with pSANF and pT-EF5 measured after 48 hours of culture was shown in Table 5.
Table 5












TABLE 5







Dry Cell Weight
Yield
Content
Productivity



(gDCW/L)
(mg/L)
(mg/gDCW)
(mg/L/hr)









5.19
134.1
25.8
2.79










EXAMPLE 7
Evaluation of Lycopene Productivity of E. coli Transformed with the Vector which Contains the Combination of the crtE Gene from Rhodobacter sphaeroides (Sequence 7) and the crtB and crtI Genes from Sea Metagenome with Mevalonate Synthesis Genes
The crtE gene in the vector pT5-LYC-idi of the Example 2 was substituted by a same gene from Rhodobacter sphaeroides to construct recombinant vector pT-RF5 (FIG. 7), which was transformed into E. coli to evaluate lycopene productivity by the same method of Example 3.
The lycopene productivity of the E. coli transformed with pSANF and pT-RF5 was shown in Table 6.
Table 6












TABLE 6







Dry Cell Weight
Yield
Content
Productivity



(gDCW/L)
(mg/L)
(mg/gDCW)
(mg/L/hr)









5.63
106.4
18.9
2.22










EXAMPLE 8
Evaluation of Lycopene Productivity of E. coli Transformed with the Vector which Contains the Combination of the crtE Gene from Synechocystis sp. PCC6803 (Sequence 8) and the crtB and crtI Genes from Sea Metagenome with the Mevalonate Synthesis Genes
The crtE gene in the vector pT5-LYC-idi of the Example 2 was substituted by a same gene from Synechocystis sp. PCC6803 to construct recombinant vector pT-SF5 (FIG. 8), which was transformed into E. coli to evaluate lycopene productivity by the same method of Example 3.
The lycopene productivity of the E. coli transformed with pSANF and pT-SF5 was shown in Table 7.
Table 7












TABLE 7







Dry Cell Weight
Yield
Content
Productivity



(gDCW/L)
(mg/L)
(mg/gDCW)
(mg/L/hr)









4.52
158.2
35.0
3.30










EXAMPLE 9
Evaluation of Lycopene Productivity of the Fed-Batch Cultured E. coli Transformed with the Vector which Contains the Combination of the crtE Gene from Synechocystis sp. PCC6803 and the crtB and crtI Genes from Sea Metagenome with Mevalonate Synthesis Genes
The E. coli transformed with the pSANF and pT-SF5 of Example 8 was subjected to the fed-batch culture.
A Biostat B fermenter (B. Brown Biotech International) was used for the fed-batch culture. The total volume of the medium container was 5 L, and the working volume was 2 L.
The fermenter was equipped with a pH meter to monitor proton concentration, a DO meter to monitor the concentration of dissolved oxygen, and a foam sensor which can detect foams in order to remove the foams appearing during the fermentation.
During the fermentation, pH was controlled with ammonia solution and 30% phosphoric acid, and the compressed air being injected into the fermenter was controlled to maintain the flow rate of 2 L/min. The temperature of the fermenter was kept at 30° C., while appropriate maintenance was made to keep the shaking speed between 200 rpm and 1,000 rpm and the concentration of dissolved oxygen between 20% and 30%. The fermenter contained 2 L of sterilized culture median for lycopene production. The culture median used for lycopene production was the modified Kortz culture median (D. J. Korz et al., J. of Biotechnology, 39, 59-65, 1995), whose pH was adjusted to 7.0 prior to autoclaving, and whose composition was as follows: glycerol 2%, KH2PO4 1.33%, (NH4)2HPO4 0.4%, MgSO4.7H2O 0.12%, citric acid 0.17%, EDTA 8.4 mg/L, CoCl2.6H2O 2.5 mg/L, MnCl2.4H2O 15.0 mg/L, CuCl2.2H2O 1.5 mg/L, H2BO3 3.0 mg/L, Na2MoO4.2H2O 2.5 mg/L, Zn(CH3COO)2.2H2O 13.0 mg/L, and Fe(III) citrate 100 mg/L. The fermenter containing the culture medium was autoclaved at 121° C. for 15 minutes to sterilize the fermenter of any microorganism inside, and then the antibiotics ampicillin and chloramphenicol were added to the concentrations of 50 ppm and 20 ppm, respectively, prior to inoculation.
The colony of Example 8 was inoculated into 3 mL of LB medium in a test tube using a platinum loop, followed by the first preincubation in a shaking fermenter at 30° C. When the optical density (hereafter O.D.) reached around 1.0 during the culture, as measured by a spectrophotometer at 600 nm, a 2 mL aliquot of the culture medium was taken and inoculated into 100 mL of fresh culture medium in a 500 mL Erlenmeyer flask. The inoculated culture medium was subjected to the second preincubation at 30° C., which was then, when the O.D. reached 1.0 again, inoculated into the whole medium in the autoclaved fermenter for the fed-batch culture. During the culture, whenever carbon sources were depleted, nutrients were supplied by adding 0.4% of culture medium for each supply; the culture medium was composed of 70% glycerol and 2% MgSO4.7H2O.
At several time points with regular intervals during the culture, an aliquot of the culture medium was taken to measure the O.D. and the yield (mg/L). The lycopene productivity measured after 48 hours of culture was shown in Table 8.
Table 8












TABLE 8







Dry Cell Weight
Yield
Content
Productivity



(gDCW/L)
(mg/L)
(mg/gDCW)
(mg/L/hr)









39.8
466
11.7
9.7










EXAMPLE 10
Enhancement of Lycopene Productivity by IPTG Induction
The colony of Example 8 was inoculated into 3 mL of LB medium in a test tube using a platinum loop, followed by the first preincubation in a shaking fermenter at 30° C. When the O.D. reached around 1.0 during the culture, as measured by a spectrophotometer at 600 nm, a 2 mL aliquot of the culture medium was taken and inoculated into 100 mL of fresh culture medium in a 500 mL Erlenmeyer flask. The inoculated culture medium was subjected to the second preincubation at 30° C., which was then, when the O.D. reached 1.0 again, inoculated into the whole medium in the autoclaved fermenter for the fed-batch culture by the same method as in Example 9.
At several time points with regular interval during the culture, an aliquot of the culture medium was taken to measure the O.D. and the yield (mg/L). When the O.D. reached 70.0, as measured by a spectrophotometer at 600 nm, IPTG induction was carried out at concentration of 0.025 mM. The lycopene productivity measured after 48 hours of culture was shown in Table 9.
Table 9












TABLE 9







Dry Cell Weight
Yield
Content
Productivity



(gDCW/L)
(mg/L)
(mg/gDCW)
(mg/L/hr)









46.1
1754
38.1
36.5










The crtE, crtB and crtI genes may be used for lycopene production either alone or through the combination of at least one of them with previously known genes; while the recombinant strain which contains the combination of genes of the present invention was able to give improved productivity compared to that of previously known inventions. In addition, the lycopene productivity could be further enhanced by introducing the genes required for the mevalonate pathway so that lycopene could be produced via the mevalonate pathway in E. coli. Therefore, the present invention can be very usefully applied to mass production of lycopene, for which there are increasing needs recently due to its antioxidant activity and the anticancer effect.
Sequence Listing
Sequence 1 is the nucleotide sequence of the gene crtE (867 bp) from sea metagenome.
Sequence 2 is the amino acid sequence of geranylgeranyl pyrophosphate synthase (288 amino acids) encoded by the gene crtE.
Sequence 3 is the nucleotide sequence of the gene crtB (909 bp) from sea metagenome.
Sequence 4 is the amino acid sequence of phytoene synthase (302 amino acids) encoded by the gene crtB.
Sequence 5 is the nucleotide sequence of the gene crtI (1,485 bp) from sea metagenome.
Sequence 6 is the amino acid sequence of phytoene desaturase (494 amino acids) encoded by the gene crtI.
Sequence 7 is the nucleotide sequence of the gene crtE of Rhodobacter sphaeroides. 
Sequence 8 is the nucleotide sequence of the gene crtE of Synechocystis sp. PCC 6803.

## Claims

1. A method of producing lycopene comprising:
preparing a recombinant vector containing genes encoding proteins, which are required for lycopene biosynthesis, wherein the genes involved in lycopene biosynthesis are one or more of crtE with the nucleotide SEQ ID NO.: 1, crtB with the nucleotide SEQ ID NO.:3 and crtI with the nucleotide SEQ ID NO.:5;
transforming the recombinant vector into E. coli; and
culturing the E. coli transformant and recovering lycopene from the culture medium.

2. The method of claim 1, wherein the recombinant vector further comprises one or more of mvaK1, mvaD, mvaK2, mvaE, mvaS and idi in addition to the genes involved in lycopene biosynthesis.

3. The method of claim 2, wherein the idi gene is idi of E. coli.

4. The method of, claim 1 wherein the transformant is cultured under the following conditions: culture temperature ranging from 25° C. to 35° C., pH of culture medium before autoclaving ranging from 7.0 to 7.5, shaking speed ranging from 200 rpm to 1,000 rpm, and the concentration of dissolved oxygen ranging from 20% to 30%.

5. The method of claim 1 wherein the transformant is cultured by a fed-batch culture for mass production of lycopene.