Patent Publication Number: US-2022228177-A1

Title: Recombinant microorganism having high ability to produce lutein and method for producing lutein using the same

Description:
TECHNICAL FIELD 
     The present invention relates to a recombinant microorganism having high ability to produce lutein and a method of producing lutein using the same, and more specifically, to a recombinant microorganism having ability to produce lutein, which is obtained by introducing a lutein biosynthetic pathway into a host cell having ability to produce farnesyl diphosphate (FPP). 
     BACKGROUND ART 
     Lutein is one of the xanthophylls naturally found in egg yolks, fruits, and green leafy vegetables. Lutein is abundant in the macula of the human eye and functions to provide protection against oxidative stress and radiation. Consuming such lutein can prevent macular degeneration and cataracts, protect the skin from UV rays, and help prevent cancer and cardiovascular disease. Due to these effects, the demand and market for lutein are increasing. 
     Lutein that is supplied to the market is mainly extracted from marigold flowers, but the process of purifying lutein from the extract is complicated because marigold flowers also produce esterified lutein which is difficult to separate from lutein. Since the chemical structure of lutein is asymmetrical and various lutein isomers exist, chemical synthesis of lutein is also inefficient. Efforts have been made to overproduce lutein by manipulating the genes of plants and microalgae, but the lutein production and productivity have not met expectations. 
     For example, U.S. Pat. No. 5,530,189 describes a recombinant plant obtained by introducing crtE, crtB and crtl genes into a plant and producing an increased amount of lutein, but does not report on a recombinant microorganism having ability to produce lutein. U.S. Pat. No. 10,059,974 describes a recombinant microorganism having ability to produce lutein, which is obtained by introducing genes encoding CYP97A, CYP97B and CYP97C proteins and genes encoding lycopene cyclase proteins, but has a disadvantage in that lutein is produced at a rate similar to or lower than beta-carotene. 
     Korean Patent No. 10-1339686 describes a method for producing chlorella with a high lutein content, but this method is a method of increasing the content by modifying the culture conditions rather than modifying the metabolic pathway, and has a disadvantage in that the content is not so high. Korean Patent No. 10-2019448 describes a novel microalga with high lutein productivity, but has a disadvantage that the microalga is only a newly discovered strain. 
     Accordingly, the present inventors have made extensive efforts to solve the above-described problems and develop a strain capable of producing a large amount of lutein, and as a result, have found that, when a lutein biosynthetic pathway is introduced into a host cell having ability to produce farnesyl diphosphate, it is possible to produce a recombinant microorganism having ability to produce lutein, and when a substrate tunnel is constructed in the recombinant microorganism and an electron tunnel is produced in the recombinant microorganism and then the intracellular production of C5 heme is increased, the recombinant microorganism is capable of producing a large amount of lutein, thereby completing the present invention. 
     SUMMARY OF THE INVENTION 
     An object of the present invention is to provide a recombinant strain capable of producing a large amount of lutein. 
     Another object of the present invention is to provide a method of producing lutein using the strain. 
     In order to achieve the above object, the present invention provides a recombinant microorganism having ability to produce lutein, which is obtained by introducing a lutein biosynthetic pathway into a host cell having ability to produce farnesyl diphosphate (FPP). 
     The present invention also provides a recombinant microorganism having enhanced ability to produce lutein, which is obtained by introducing a lutein biosynthetic pathway into a host cell having farnesyl diphosphate (FPP) and introducing any one or more selected from the group consisting of substrate tunnel formation, electron tunnel formation, and C5 heme production pathway modification. 
     The present invention also provides a method for producing lutein comprising steps of: (a) producing lutein by culturing the recombinant microorganism; and (b) recovering the produced lutein. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  shows the major metabolic pathways for lutein biosynthesis and the results of flask culture.  FIG. 1( a )  shows enzymes involved in an exogenous lutein biosynthetic pathway constructed in a cell according to the present invention. Bent arrows and the character T indicate promoters and transcription terminators, respectively. The abbreviations indicate the following: 5-ALA, 5-aminolevulinic acid; DXP, 1-deoxy-D-xylulose 5-phosphate; FPP, farnesyl diphosphate; L-Glu-tRNA, L-glutamyl-tRNA; G3P, D-glyceraldehyde 3-phosphate; GGPP, geranylgeranyl diphosphate; and TCA cycle, tricarboxylic acid cycle.  FIG. 1( b )  shows the results of flask culture of LUT1 to 4 and LUT4M strains.  FIG. 1( c )  schematically shows electron transfer from ATR2 to two P450s. It was expected that, when the CipB scaffold proteins linked to ATR2, trLUT5 and trLUT1 were brought together in the cell, electron transfer would occur more efficiently due to the closer distance therebetween.  FIG. 1( d )  shows the results of flask culture of LUT4M and LUT5M strains. Error bars mean standard deviation (n=3). **P&lt;0.01; ***P&lt;0.001. The abbreviations mean the following: ns, not significant. 
         FIG. 2  shows increased lutein production resulting from increased heme supply and culture condition optimization and the results of fed-batch culture of LUT5MH1.  FIG. 2( a )  shows the biosynthetic pathway of lutein and heme, and electron transfer from NADPH to oxygen via ATR2 and trLUT5/trLUT1. The abbreviations mean the following: 5-ALA, 5-aminolevulinic acid; DXP, 1-deoxy-D-xylulose 5-phosphate; L-Glu-tRNA, L-glutamyl-tRNA; G3P, D-glyceraldehyde 3-phosphate; and TCA cycle, tricarboxylic acid cycle.  FIG. 2( b )  shows the results of flask culture of LUT5M, LUT5MH1, and LUT5MH2 strains.  FIG. 2( c )  shows the result of comparison between R/2 medium and MR medium. The LUT5MH1 strain was cultured in R/2 or MR medium at 30° C., and then gene expression was induced with 1 mM IPTG.  FIG. 2( d )  shows the results of testing various culture temperatures for lutein production. The LUT5MH1 strain was cultured in R/2 medium at each of 37, 30, 28, 25 and 22° C., and gene expression was induced using 1 mM IPTG.  FIG. 2( e )  shows the results of comparing the effect of IPTG concentration, used for gene expression, on lutein production. LUT5MH1 was cultured in R/2 medium at 28° C., and when the OD 600  reached 0.4 to 0.6, IPTG was added to final concentrations of 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 mM. Error bars mean standard deviation (n=3). *P&lt;0.05; **P &lt;0.01; ***P&lt;0.001. The abbreviations mean the following: ns, significant.  FIG. 2( f )  depicts photographs of the incubator during fed-batch culture of LUT5MH1.  FIG. 2( g )  shows the results of fed-batch culture of LUT5MH1. The culture temperature was maintained at 37° C. and then decreased to 28° C. after addition of 0.5 mM IPTG. In addition, carbon starvation was induced for 2 hours (at 12 to 14 hours) after depletion of the initially supplied carbon source. 
         FIG. 3  shows the results of analysis of the intracellular localization of enzymes linked to CipA or CipB, the intracellular formation of protein crystalline inclusions (PCIs), and verification of lutein production.  FIG. 3( a )  shows the HPLC-MS chromatograms and MS spectra of the lutein standard (molecular weight: 568.89 g mol −1 ) and the LUT3 cell extract. The arrows indicate the lutein peaks corresponding to the MS spectra (m/z=551.4 [M+H-H20] - 1. FIG.  3  ( b ) shows micrographs of WLGB-RPP strains having one or two of LUT1, pLUT2, pLUT3, pLUT4, pLUT5, pLUT6 and pLUT7, respectively. Black arrows indicate PCIs formed within cells. 
         FIG. 3( c )  shows the results of SDS-PAGE performed to analyze the intracellular localization of ATR2, trLUT5 and trLUT1. Each lane corresponds to cytoplasmic protein (lane 1) and membrane protein (lane 2) of WLGB-RPP (pTrc99a), and cytoplasmic protein (lane 3) and (4) membrane protein (lane 4) of WLGB-RPP (pLUT3). The arrows in lane 3 indicate trLUT5 (63.82 kDa) and trLUT1 (56.69 kDa) in order from the top, and the arrow in lane 4 indicates ATR2 (78.91 kDa).  FIG. 3( d )  shows the results of SDS-PAGE analysis of PCIs formed by CipA. PCIs were isolated from strains having WLGB-RPP strains having pTac15k and pBBR1Tac (lane 1), pLUT1 and pLUT2 (lane 2), pLUT4 and pLUT5 (lane 3), pLUT4 and pLUT6 (lane 4), and pLUT1 and pLUT5 (lane 5), respectively, and loaded in the corresponding lanes. The arrow indicates CipA-CrtI (66.74 kDa).  FIG. 3( e )  shows the results of SDS-PAGE analysis of PCIs formed by CipB. PCIs were isolated from WLGB-RPP strains having pTrc99a (lane 1), pLUT3 (lane 2) and pLUT7 (lane 3), respectively, and loaded in the corresponding lanes. It appears that proteins in lane 2 include membrane proteins including ATR2 (78.91 kDa), as well as trLUT5 (63.82 kDa) and trLUT1 (56.69 kDa), which are originally cytoplasmic proteins, but are prematurely terminated, aggregated, or misfolded. The arrows in lane 3 indicate CipA-ATR2 (90.19 kDa), CipA-trLUT5 (75.09 kDa) and CipA-trLUT1 (67.96 kDa) in order from the top. Lane (L) in all SDS-PAGE results corresponds to protein size markers. 
         FIG. 4  shows the results of fed-batch culture of the LUT5MH1 strain under different temperature conditions and different IPTG concentration conditions. The culture temperature was maintained at 28° C. ( FIG. 4 a   ) or  37 ° C. ( FIG. 4 b   ), and then decreased to 28° C. after addition of 0.05 mM IPTG. For other graphs, the culture temperature was maintained at 37° C., and then decreased to 28° C. after addition of 0.2 mM ( FIG. 4 c   ), 0.5 mM ( FIG. 4 d  and 4 f   ), or 1 mM IPTG ( FIG. 4 e   ). In the fed-batch culture in  FIG. 4( f ) , carbon starvation was induced for 2 hours after depletion of the initial carbon source. 
     
    
    
     DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF THE INVENTION 
     Unless otherwise defined, all technical and scientific terms used in the present specification have the same meanings as commonly understood by those skilled in the art to which the present disclosure pertains. In general, the nomenclature used in the present specification is well known and commonly used in the art. 
     Definitions of key terms used in the detailed description of the present invention, etc. are as follows. 
     As used herein, the term “host cell” means any cell capable of expressing a functional gene and/or gene product derived from another cell or organ. 
     As used herein, the term “gene” is to be considered in its broadest sense, and the gene may encode a structural protein or a regulatory protein. In this case, examples of the regulatory protein include transcription factors, heat shock proteins, or proteins involved in DNA/RNA replication, transcription and/or translation. 
     As used herein, the term “lutein” refers to a compound having a molecular structure of Structural Formula 1 below. 
     
       
         
         
             
             
         
       
     
     As used herein, the term “weakening” is meant to encompass reducing the activity of an enzyme, which is encoded by the gene of interest, by mutation, substitution or deletion of one or more nucleotides in the gene or by introduction of one or more nucleotides into the gene, and includes blocking a part or a significant part of a biosynthetic pathway in which the enzyme encoded by the gene is involved. 
     As used herein, the term “lacking” is meant to encompass preventing the gene of interest from being expressed or from showing enzymatic activity even if expressed, by mutation, substitution or deletion of some or all of the nucleotides in the gene or by introduction of some nucleotides into the gene, and includes blocking a biosynthetic pathway in which the enzyme encoded by the gene is involved. 
     As used herein, the term “amplification” is meant to encompass increasing the activity of an enzyme, which is encoded by the gene of interest, by mutation, substitution or deletion of one or more nucleotides in the gene, or introduction of one or more nucleotides into the gene, or introduction of a gene derived from another microorganism, which encodes the same enzyme. 
     In the present invention, examination was made to determine whether lutein was produced when a lutein biosynthetic pathway was introduced into an existing host cell having ability to produce farnesyl diphosphate. In addition, in order to develop a strain capable of producing lutein in large amounts by manipulating various metabolic pathways in the microorganism into which the lutein biosynthetic pathway has been introduced, examination was made, and as a result, it was confirmed that, when a substrate tunnel, electron tunnel, and C5 heme production pathway in the microorganism were manipulated, the microorganism could produce a large amount of lutein. 
     That is, in one example of the present invention, a lutein biosynthetic pathway was introduced into a WLGB-RPP strain having ability to produce lycopene (Choi, H S, et al., Appl Environ Microb 76, 3097-3105, 2010). Specifically, a recombinant  E. coli  platform strain having ability to produce lutein was constructed by inserting genes encoding enzymes derived from bacteria or eukaryotes. 
     In addition, various metabolic pathways in the  E. coli  platform strain were manipulated. There are two representative bottlenecks in the lutein biosynthetic pathway: promiscuous enzymes that are involved in metabolic flux; and two cytochrome P450 enzymes that are generally less active when expressed in microorganisms. 
     First, the problem caused by promiscuous enzymes was overcome by minimizing by-products through the creation of a substrate tunnel. The problem caused by P450 may occur due to the inefficiency of electron transfer between P450 and reductase, and taking this into consideration, an electron tunnel was created to facilitate electron transfer between the two enzymes. 
     In addition, when the intracellular production of heme, which is a cofactor of P450 acting as a mediator of electron transfer, was increased, P450 activity was further increased, thereby significantly increasing the production of lutein. 
     More specifically, it was confirmed that, when crtE, crtB, crtI, cipA-trLUT2, cipA-trLCYBmut, cipB-trLUT5, cipB-trLUT1, cipB-ATR2, hemA fbr  and hemL genes were introduced into a WLGB-RPP (ΔlacI ΔgdhA ΔgpmB ptrc dxs idi ispA pps) strain, the strain could produce lutein with the highest productivity ( FIGS. 1 and 2 ). 
     Therefore, in one aspect, the present invention is directed to a recombinant microorganism having ability to produce lutein, which is obtained by introducing a lutein biosynthetic pathway into a host cell having ability to produce farnesyl diphosphate (FPP). 
     In the present invention, the host cell having ability to produce farnesyl diphosphate (FPP) may be a host cell which lacks at least one gene selected from the group consisting of lad (lactose operon repressor) gene, gdhA (NADP-specific glutamate dehydrogenase) gene and gpmB (phosphoglycerate mutase) gene and in which at least one gene selected from the group consisting of dxs (1-deoxyxylulose-5-phosphate synthase) gene, idi (isopentenyl diphosphate (IPDP) isomerase) gene, ispA (geranyltranstransferase/dimethylallyltranstransferase) gene and pps (PEP synthase) gene has been introduced or amplified. 
     More preferably, the host cell may be a WLGB-RPP strain, without being limited thereto. 
     In the present invention, the lutein biosynthetic pathway may be a lutein biosynthetic pathway into which at least one gene selected from the group consisting of crtE (geranylgeranyl pyrophosphate synthase) gene, crtB (phytoene synthase) gene, crtI (phytoene dehydrogenase) gene, LUT2 (lycopene ε-cyclase) gene, LCYB (lycopene β-cyclase) gene, ATR2 (cytochrome P450 reductase) gene, LUT5 (β-carotene 3-hydroxylase) gene and LUT1 (carotene ε-monooxygenase) gene has been introduced. 
     In the present invention, the LUT2 gene may be represented by the nucleotide sequence of SEQ ID NO: 19, without being limited thereto. 
     In the present invention, the LCYB gene may be represented by the nucleotide sequence of SEQ ID NO: 20, without being limited thereto. 
     In the present invention, the LUT5 gene may be represented by the nucleotide sequence of SEQ ID NO: 21, without being limited thereto. 
     In the present invention, the LUT1 gene may be represented by the nucleotide sequence of SEQ ID NO: 22, without being limited thereto. 
     In the present invention, the LCYB gene may encode G451E mutant protein. 
     In the present invention, when the LCYB gene encodes the G451E mutant protein, it may be represented by the nucleotide sequence of SEQ ID NO: 58, without being limited thereto. 
     In the present invention, the recombinant microorganism into which the lutein biosynthetic pathway has been introduced may be a recombinant microorganism into which any one or more selected from the group consisting of substrate tunnel formation, electron tunnel formation and C5 heme production pathway modification has been introduced so that the ability to produce lutein is further enhanced. 
     In the present invention, the substrate tunnel formation may be performed by introducing the cipA gene. 
     In the present invention, the introduction of the cipA gene may comprise modifying any one or more genes, selected from the group consisting of crtl, LUT2 and LCYB, into any one or more genes selected from the group consisting of cipA-crtl, cipA-LUT2 and cipA-LCYB. 
     In the present invention, the cipA-crtl gene may be represented by the nucleotide sequence of SEQ ID NO: 33, without being limited thereto. 
     In the present invention, the cipA-LUT2 gene may be represented by the nucleotide sequence of SEQ ID NO: 34, without being limited thereto. 
     In the present invention, the cipA-LCYB gene may be represented by the nucleotide sequence of SEQ ID NO: 35, without being limited thereto. 
     In the present invention, when the cipA-LCYB gene encodes a G451E mutant protein, it may be represented by the nucleotide sequence of SEQ ID NO: 36, without being limited thereto. 
     In the present invention, the electron tunnel formation may be performed by introducing the cipB gene. 
     In the present invention, the introduction of the cipB gene may comprise modifying any one or more genes, selected from the group consisting of ATR2, LUT5 and LUT1, into any one or more genes selected from the group consisting of cipB-ATR2, cipB-LUT5 and cipB-LUT1. 
     In the present invention, the cipB-ATR2 gene may be represented by the nucleotide sequence of SEQ ID NO: 41, without being limited thereto. 
     In the present invention, the cipB-LUT5 gene may be represented by the nucleotide sequence of SEQ ID NO: 42, without being limited thereto. 
     In the present invention, the cipB-LUT1 gene may be represented by the nucleotide sequence of SEQ ID NO: 43, without being limited thereto. 
     In the present invention, the C5 heme production pathway modification may be performed by introducing any one or more genes selected from the group consisting of hemA, hemL, hemB and hemH. 
     In the present invention, the hemA gene may encode a mutant protein that is resistant to feedback inhibition. 
     In the present invention, when the hemA gene encodes a mutant protein that is resistant to feedback inhibition, it may be represented by the nucleotide sequence of SEQ ID NO: 56, without being limited thereto. 
     In the present invention, the hemL gene may be represented by the nucleotide sequence of SEQ ID NO: 57, without being limited thereto. 
     As used herein, the term “vector” refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself. In the present specification, “plasmid” and “vector” are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. For the purposes of the present invention, a plasmid vector is preferably used. A typical plasmid vector that may be used for these purposes has a structure including: (a) a replication origin that allows effective replication so as to include several to hundreds of plasmid vectors per host cell; (b) an antibiotic resistance gene that enables selection of a host cell transformed with the plasmid vector; and (c) a restriction enzyme cleavage site into which a foreign DNA fragment may be inserted. Even if an appropriate restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or a linker according to a conventional method. Even if an appropriate restriction enzyme cleavage site not present, the vector and the foreign DNA may be easily ligated using a synthetic oligonucleotide adapter or a linker according to a conventional method. After ligation, the vector is required to be transformed into the appropriate host cell. The transformation may be easily achieved by a calcium chloride method or electroporation (Neumann, et al.,  EMBO J.,  1:841, 1982). 
     In the present invention, the nucleotide sequence is “operably linked” when it is placed in a functional relationship with another nucleic acid sequence. The term “operably linked” means that a gene and one or more transcriptional regulatory sequences are connected in such a way to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking between these sequences is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. 
     In the present invention, the host cell may be selected from the group consisting of  E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Cyanobacterium, and Cyclobacterium.    
     In another aspect, the present invention is directed to a recombinant microorganism having enhanced ability to produce lutein, which is obtained by introducing a lutein biosynthetic pathway into a host cell having ability to produce farnesyl diphosphate (FPP) and introducing at least one selected from the group consisting of substrate tunnel formation, electron tunnel formation, and C5 heme production pathway modification. 
     In the present invention, the host cell having ability to produce farnesyl diphosphate (FPP) may be a host cell which lacks at least one gene selected from the group consisting of lad (lactose operon repressor) gene, gdhA (NADP-specific glutamate dehydrogenase) gene and gpmB (phosphoglycerate mutase) gene and in which at least one gene selected from the group consisting of dxs (1-deoxyxylulose-5-phosphate synthase) gene, idi (isopentenyl diphosphate (IPDP) isomerase) gene, ispA (geranyltranstransferase/dimethylallyltranstransferase) gene and pps (PEP synthase) gene has been introduced or amplified. 
     More preferably, the host cell may be a WLGB-RPP strain, without being limited thereto. 
     In the present invention, the lutein biosynthetic pathway may be a lutein biosynthetic pathway into which at least one gene selected from the group consisting of crtE (geranylgeranyl pyrophosphate synthase) gene, crtB (phytoene synthase) gene, crtI (phytoene dehydrogenase) gene, LUT2 (lycopene ε-cyclase) gene, LCYB (lycopene β-cyclase) gene, ATR2 (cytochrome P450 reductase) gene, LUT5 (β-carotene 3-hydroxylase) gene and LUT1 (carotene ε-monooxygenase) gene has been introduced. 
     In the present invention, the LUT2 gene may be represented by the nucleotide sequence of SEQ ID NO: 19, without being limited thereto. 
     In the present invention, the LCYB gene may be represented by the nucleotide sequence of SEQ ID NO: 20, without being limited thereto. 
     In the present invention, the LUT5 gene may be represented by the nucleotide sequence of SEQ ID NO: 21, without being limited thereto. 
     In the present invention, the LUT1 gene may be represented by the nucleotide sequence of SEQ ID NO: 22, without being limited thereto. 
     In the present invention, the LCYB gene may encode G451E mutant protein. 
     In the present invention, when the LCYB gene encodes the G451E mutant protein, it may be represented by the nucleotide sequence of SEQ ID NO: 58, without being limited thereto. 
     In the present invention, the substrate tunnel formation may be performed by introducing the cipA gene. 
     In the present invention, introduction of the cipA gene may comprise modifying any one or more genes, selected from the group consisting of crtl, LUT2 and LCYB, into any one or more genes selected from the group consisting of cipA-crtl, cipA-LUT2 and cipA-LCYB. 
     In the present invention, the cipA-crtl gene may be represented by the nucleotide sequence of SEQ ID NO: 33, without being limited thereto. 
     In the present invention, the cipA-LUT2 gene may be represented by the nucleotide sequence of SEQ ID NO: 34, without being limited thereto. 
     In the present invention, the cipA-LCYB gene may be represented by the nucleotide sequence of SEQ ID NO: 35, without being limited thereto. 
     In the present invention, when the cipA-LCYB gene encodes a G451E mutant protein, it may be represented by the nucleotide sequence of SEQ ID NO: 36, without being limited thereto. 
     In the present invention, the electron tunnel formation may be performed by introducing the cipB gene. 
     In the present invention, the introduction of the cipB gene may comprise modifying any one or more genes, selected from the group consisting of ATR2, LUT5 and LUT1, into any one or more genes selected from the group consisting of cipB-ATR2, cipB-LUT5 and cipB-LUT1. 
     In the present invention, the cipB-ATR2 gene may be represented by the nucleotide sequence of SEQ ID NO: 41, without being limited thereto. 
     In the present invention, the cipB-LUT5 gene may be represented by the nucleotide sequence of SEQ ID NO: 42, without being limited thereto. 
     In the present invention, the cipB-LUT1 gene may be represented by the nucleotide sequence of SEQ ID NO: 43, without being limited thereto. 
     In the present invention, the C5 heme production pathway modification may be performed by introducing any one or more genes selected from the group consisting of hemA, hemL, hemB and hemH. 
     In the present invention, the hemA gene may encode a mutant protein that is resistant to feedback inhibition. 
     In the present invention, when the hemA gene encodes a mutant protein that is resistant to feedback inhibition, it may be represented by the nucleotide sequence of SEQ ID NO: 56, without being limited thereto. 
     In the present invention, the hemL gene may be represented by the nucleotide sequence of SEQ ID NO: 57, without being limited thereto. 
     In still another aspect, the present invention is directed to a method for producing lutein comprising steps of: (a) producing lutein by culturing the recombinant microorganism; and (b) recovering the produced lutein. 
     In the present invention, the process of culturing the recombinant microorganism and recovering lutein may be performed using a culture method (batch culture, or fed-batch culture) and a lutein separation and purification method commonly known in a conventional fermentation process. 
     In the present invention, biotechnological production of lutein may be performed intracellularly or extracellularly (in vivo or in vitro). 
     In the present invention, step (a) may comprise a step of making the temperature of the culturing after expression of the lutein biosynthetic pathway genes different from the temperature of the culturing before the expression, and inducing carbon starvation after the expression. 
     Preferably, the step of producing lutein by culturing the recombinant microorganism may comprise the following steps, without being limited thereto. 
     (a) culturing the recombinant microorganism in R/2 medium at 37° C.; 
     (b) when the OD 600  value reaches 20 to 30, injecting IPTG to a final concentration of 0.5 mM, changing the temperature of the culturing to 28° C., and culturing the recombinant microorganism; and 
     (c) culturing the recombinant microorganism without supplying a carbon source for 2 hours after depletion of the initial carbon source. 
     In the present invention, the method may further comprise, after step (c), a step of additionally injecting a carbon source and culturing the recombinant microorganism. 
     EXAMPLES 
     Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by the following examples, and it will be apparent to those skilled in the art that various changes or modifications may be made within the idea and scope of the present invention. 
     Example 1. Construction of Lutein Production Pathway 
     1-1. Selection of Lutein Biosynthetic Pathway Genes 
     First, crtE, crtB and crtI from  Pantoea ananatis , which are genes producing lycopene, a precursor of lutein, were introduced into a pTac15k plasmid, thereby constructing pLUT1. Next, in order to convert lycopene to α-carotene, LUT2 and LCYB from  Arabidopsis thaliana , which were codon-optimized and from which the N-terminal signal peptide coding sequence was cut out, were introduced into a pBBR1Tac plasmid, thereby constructing pLUT2. The N-terminal signal peptide sequence was predicted using ChloroP software (Emanuelsson, O., Nielsen, et al.,  Protein Sci  8, 978-984, 1999). 
     Finally, in order to convert alpha-carotene into lutein, LUT5 and LUT1 from  A. thaliana , which were codon-optimized and from which the N-terminal signal peptide coding sequence was cut out, were introduced into a pTrc99a plasmid. LUT5 and LUT1 are cytochrome P450 enzymes (P450) and require a partner reductase to have activity in  E. coli . Thus, ATR2 from  A. thaliana  was codon-optimized and also introduced, thereby constructing pLUT3. ATR2 also has a putative signal peptide sequence at the N-terminus, but the signal peptide sequence was not removed, in consideration of reports that the activity was higher when the entire sequence was expressed in  E. coli .  FIG. 1 a    shows the major lutein biosynthetic pathway. 
     1-2. Plasmid Construction 
     A more detailed plasmid construction procedure is as follows. First, either DNA digestion using restriction enzymes or Gibson&#39;s method (Gibson, D. G. et al.  Nat Methods  6, 343-U341, 2009) was used to construct the plasmids in this study. crtE, crtI and crtB from  P. ananatis  were used after amplification from pCar184 (Choi, H. S., et al.,  Appl Environ Microb  76, 3097-3105, 2010), and trLUT2, trLCYB, ATR2, trLUT5 and trLUT1 genes from  A. thaliana  were synthesized and used after codon optimization. 
     To construct pLUT1, crtE and crtl-crtB from pCar184 were amplified using a combination of crtE_F/crtE_R1 primers and a combination of crtI_F1/crtB_R primers, respectively, and then ligated together by PCR and inserted into the SacI/XbaI cleavage sites of pTac15k. To construct pLUT2, trLUT2 and trLCYB genes were amplified using a combination of trLUT2_F/R1 primers and a combination of trLCYB_F/R primers, respectively, and then inserted into the SacI/XbaI and XbaI/PstI cleavage sites of the pBBR1Tac plasmid, respectively. 
     Before construction of pLUT3, pTrc99a plasmids expressing each of ATR2, trLUT5 and trLUT1 were constructed. The genes were amplified using ATR2_F1/R1, trLUT5_F/R1, and trLUT1_F/R1 primer sets, respectively, and then inserted into EcoRI/BamHI cleavage sites of pTrc99a plasmids, thereby constructing pTrc-ATR2, pTrc-trLUT5, and pTrc-trLUT1, respectively. The trLUT5 gene ligated with the trc promoter was amplified from pTrc-trLUT5 using pTrc_F1/trLUT5_R2 primers, and the trLUT1 gene ligated with the trc promoter was amplified using pTrc_F2/trLUT1_R2 primers. Then, the amplified genes were inserted into the BamHI/XbaI and XbaI/SalI cleavage sites of the pTrc-ATR2 plasmid, respectively, thereby constructing pLUT3. 
     Table 1 below shows the sequences of the primers used in plasmid construction. 
     
       
         
           
               
               
               
             
               
                 TABLE 1  
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO 
                 Name 
                 Sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 crtE_F 
                 AGACAGGAGCTCTTCACACAGGAAACAATGTATC 
               
               
                   
                   
                 CGTTTATAAGGAC 
               
               
                   
               
               
                 2 
                 crtE_R1 
                 TTAACTGACGGCAGCGAGTT 
               
               
                   
               
               
                 3 
                 crtI_F1 
                 AACTCGCTGCCGTCAGTTAATTCACACAGGAAAC 
               
               
                   
                   
                 AATGAAACCAACTACGGTAAT 
               
               
                   
               
               
                 4 
                 crtB_R 
                 AGACAGTCTAGATTAGAGCGGGCGCTGCCAGA 
               
               
                   
               
               
                 5 
                 trLUT2_F 
                 AGACAGGAGCTCTTCACACAGGAAACAATGGCAT 
               
               
                   
                   
                 CCGGCGGT 
               
               
                   
               
               
                 6 
                 trLUT2_R 
                 AGACAGTCTAGATTACACTTTCAGGTAGGTTTTG 
               
               
                   
               
               
                 7 
                 trLCYB_F 
                 AGACAGTCTAGATTCACACAGGAAACAATGGTCG 
               
               
                   
                   
                 TGTCGGGG 
               
               
                   
               
               
                 8 
                 trLCYB_R 
                 AGACAGCTGCAGTTAGTCGCGATCCTGCACTA 
               
               
                   
               
               
                 9 
                 ATR2_F1 
                 AGACAGGAATTCTTCACACAGGAAACAATGTCTT 
               
               
                   
                   
                 CTTCTTCTTCTTC 
               
               
                   
               
               
                 10 
                 ATR2_R1 
                 AGACAGGGATCCTTACCACACATCACGAAGAT 
               
               
                   
               
               
                 11 
                 trLUT5_F 
                 AGACAGGAATTCTTCACACAGGAAACAATGTTCA 
               
               
                   
                   
                 GCAGCTCAAG 
               
               
                   
               
               
                 12 
                 trLUT5_R1 
                 AGACAGGGATCCTTAAGAAAGGGCCGAGCTAA 
               
               
                   
               
               
                 13 
                 trLUT1_F 
                 AGACAGGAATTCTTCACACAGGAAACAATGAGTT 
               
               
                   
                   
                 CCATCGAAAAACC 
               
               
                   
               
               
                 14 
                 trLUT1_R1 
                 AGACAGGGATCCTTAACGTTGAGAAACTTTCA 
               
               
                   
               
               
                 15 
                 pTrc_F1 
                 AGACAGGGATCCTTGACAATTAATCATCCGG 
               
               
                   
               
               
                 16 
                 trLUT5_R2 
                 AGACAGTCTAGATTAAGAAAGGGCCGAG 
               
               
                   
               
               
                 17 
                 pTrc_F2 
                 AGACAGTCTAGATTGACAATTAATCATCCGG 
               
               
                   
               
               
                 18 
                 trLUT1_R2 
                 AGACAGGTCGACTTAACGTTGAGAAACTTTCA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2  
               
             
            
               
                   
               
               
                 Codon-optimized gene sequences 
               
            
           
           
               
               
               
            
               
                 SEQ ID 
                   
                   
               
               
                 NO 
                 Name 
                 Sequence 
               
               
                   
               
               
                 19 
                 trLUT2 
                 ATGGCATCCGGCGGTGGCAGCTCTGGCAGCGAATCTTGTGTGG 
               
               
                   
                   
                 CGGTTCGCGAAGACTTTGCCGATGAAGAAGACTTCGTCAAAGC 
               
               
                   
                   
                 CGGTGGCTCTGAAATCCTGTTTGTGCAGATGCAGCAAAACAAA 
               
               
                   
                   
                 GACATGGATGAACAAAGTAAACTGGTTGATAAACTGCCGCCGA 
               
               
                   
                   
                 TTTCCATCGGTGATGGCGCGCTGGACCTGGTTGTGATTGGTTGT 
               
               
                   
                   
                 GGTCCGGCCGGTCTGGCACTGGCAGCTGAAAGCGCCAAACTGG 
               
               
                   
                   
                 GCCTGAAAGTCGGTCTGATCGGCCCGGATCTGCCGTTTACCAAC 
               
               
                   
                   
                 AATTATGGTGTGTGGGAAGATGAATTTAATGACCTGGGCCTGC 
               
               
                   
                   
                 AAAAATGCATTGAACATGTGTGGCGTGAAACGATCGTTTATCTG 
               
               
                   
                   
                 GATGACGATAAACCGATTACCATTGGTCGTGCGTACGGCCGTGT 
               
               
                   
                   
                 GAGCCGTCGCCTGCTGCACGAAGAACTGCTGCGTCGCTGTGTTG 
               
               
                   
                   
                 AATCAGGTGTCTCGTATCTGAGTTCCAAAGTTGATAGTATTACG 
               
               
                   
                   
                 GAAGCATCCGATGGTCTGCGTCTGGTCGCTTGTGACGATAACAA 
               
               
                   
                   
                 TGTGATTCCGTGTCGCCTGGCTACCGTGGCGTCAGGTGCCGCCT 
               
               
                   
                   
                 CGGGTAAACTGCTGCAATATGAAGTTGGTGGCCCGCGTGTCTGT 
               
               
                   
                   
                 GTGCAAACCGCGTATGGTGTTGAAGTCGAAGTGGAAAACTCAC 
               
               
                   
                   
                 CGTACGACCCGGATCAGATGGTTTTTATGGATTATCGTGACTAC 
               
               
                   
                   
                 ACCAATGAAAAAGTCCGCAGCCTGGAAGCGGAATATCCGACCT 
               
               
                   
                   
                 TCCTGTACGCCATGCCGATGACGAAATCACGCCTGTTTTTCGAA 
               
               
                   
                   
                 GAAACCTGTCTGGCCTCGAAAGATGTGATGCCGTTTGACCTGCT 
               
               
                   
                   
                 GAAAACCAAACTGATGCTGCGTCTGGATACGCTGGGCATTCGC 
               
               
                   
                   
                 ATCCTGAAAACCTATGAAGAAGAATGGTCATACATTCCGGTTG 
               
               
                   
                   
                 GTGGCTCGCTGCCGAACACGGAACAGAAAAATCTGGCATTTGG 
               
               
                   
                   
                 TGCAGCTGCGAGCATGGTCCATCCGGCTACCGGCTATTCTGTTG 
               
               
                   
                   
                 TCCGTAGTCTGTCCGAAGCCCCGAAATACGCAAGTGTTATTGCT 
               
               
                   
                   
                 GAAATCCTGCGTGAAGAAACCACGAAACAGATTAACAGCAATA 
               
               
                   
                   
                 TCTCTCGCCAAGCGTGGGATACCCTGTGGCCGCCAGAACGTAA 
               
               
                   
                   
                 ACGCCAGCGTGCGTTTTTCCTGTTTGGTCTGGCCCTGATTGTGC 
               
               
                   
                   
                 AATTCGATACGGAAGGCATCCGCAGCTTTTTCCGTACCTTTTTC 
               
               
                   
                   
                 CGCCTGCCGAAATGGATGTGGCAGGGTTTTCTGGGCAGCACCCT 
               
               
                   
                   
                 GACGTCTGGCGATCTGGTTCTGTTTGCACTGTATATGTTCGTCAT 
               
               
                   
                   
                 TAGCCCGAACAATCTGCGCAAAGGTCTGATTAACCACCTGATCT 
               
               
                   
                   
                 CTGATCCGACCGGCGCTACGATGATCAAAACCTACCTGAAAGT 
               
               
                   
                   
                 GTAA 
               
               
                   
               
               
                 20 
                 trLCYB 
                 ATGGTCGTGTCGGGGAGCGCGGCCTTGCTGGATTTAGTCCCCGA 
               
               
                   
                   
                 AACCAAGAAAGAGAACCTGGACTTTGAGTTACCCCTTTATGAT 
               
               
                   
                   
                 ACGAGTAAATCCCAAGTTGTCGATCTGGCAATCGTCGGAGGGG 
               
               
                   
                   
                 GCCCGGCAGGGTTAGCAGTGGCCCAGCAAGTAAGTGAGGCCGG 
               
               
                   
                   
                 TCTTAGCGTGTGCTCGATTGACCCCAGTCCCAAGTTGATCTGGC 
               
               
                   
                   
                 CCAACAATTATGGAGTATGGGTCGATGAATTTGAAGCTATGGA 
               
               
                   
                   
                 CCTGTTAGACTGCCTGGATACTACTTGGAGTGGAGCAGTTGTGT 
               
               
                   
                   
                 ATGTAGATGAAGGAGTTAAAAAAGATCTGTCACGTCCTTATGG 
               
               
                   
                   
                 ACGTGTAAACCGCAAACAGTTAAAGTCAAAGATGCTGCAAAAA 
               
               
                   
                   
                 TGTATCACGAACGGTGTGAAGTTTCATCAGTCCAAAGTGACCA 
               
               
                   
                   
                 ATGTAGTTCACGAGGAGGCAAATTCAACGGTTGTCTGTAGCGA 
               
               
                   
                   
                 CGGTGTGAAAATCCAAGCCAGCGTAGTCCTGGATGCGACGGGG 
               
               
                   
                   
                 TTTTCGCGTTGCCTGGTTCAATACGACAAGCCCTACAACCCAGG 
               
               
                   
                   
                 CTACCAAGTTGCCTACGGAATTGTAGCAGAAGTAGATGGGCAC 
               
               
                   
                   
                 CCCTTCGACGTTGACAAAATGGTTTTTATGGACTGGCGTGATAA 
               
               
                   
                   
                 GCACCTGGATAGTTACCCAGAATTGAAGGAGCGCAATTCAAAA 
               
               
                   
                   
                 ATTCCTACATTCCTTTATGCGATGCCTTTCTCGTCCAATCGCATC 
               
               
                   
                   
                 TTCTTGGAGGAGACCTCCCTGGTAGCTCGCCCGGGTCTTCGCAT 
               
               
                   
                   
                 GGAAGATATCCAGGAACGCATGGCCGCTCGCTTAAAGCACTTA 
               
               
                   
                   
                 GGTATCAACGTGAAGCGTATTGAAGAAGATGAACGCTGTGTAA 
               
               
                   
                   
                 TCCCCATGGGTGGGCCACTGCCCGTGTTACCTCAACGTGTGGTA 
               
               
                   
                   
                 GGTATCGGTGGCACGGCCGGCATGGTTCATCCTAGTACCGGTTA 
               
               
                   
                   
                 CATGGTAGCCCGCACACTGGCAGCGGCACCTATTGTCGCAAAT 
               
               
                   
                   
                 GCAATCGTTCGTTATTTGGGTTCCCCCTCGTCAAACTCCTTACGC 
               
               
                   
                   
                 GGAGACCAGTTAAGTGCCGAAGTTTGGCGCGACCTTTGGCCCA 
               
               
                   
                   
                 TCGAACGTCGTCGCCAACGCGAATTCTTCTGTTTTGGGATGGAT 
               
               
                   
                   
                 ATCTTATTAAAACTGGACTTAGATGCGACCCGCCGCTTCTTCGA 
               
               
                   
                   
                 CGCATTCTTCGATCTTCAGCCACACTACTGGCACGGATTTTTAT 
               
               
                   
                   
                 CATCGCGTTTATTTCTGCCCGAGTTATTGGTATTCGGCTTGAGTC 
               
               
                   
                   
                 TTTTTTCTCATGCGTCGAACACTTCCCGCCTGGAGATCATGACT 
               
               
                   
                   
                 AAAGGCACCGTACCATTGGCTAAGATGATCAATAACTTAGTGC 
               
               
                   
                   
                 AGGATCGCGACTAA 
               
               
                   
               
               
                 21 
                 trLUT5 
                 ATGTTCAGCAGCTCAAGTAACGGCCGCGATCCACTTGAGGAAA 
               
               
                   
                   
                 ACTCAGTACCGAATGGCGTGAAAAGCCTGGAGAAATTGCAGGA 
               
               
                   
                   
                 GGAAAAACGCCGTGCTGAATTGTCGGCCCGTATCGCATCCGGT 
               
               
                   
                   
                 GCATTCACCGTTCGTAAGAGCAGCTTCCCCAGCACTGTCAAGAA 
               
               
                   
                   
                 TGGTCTTAGCAAAATCGGGATTCCTAGCAACGTATTGGATTTTA 
               
               
                   
                   
                 TGTTCGACTGGACAGGAAGTGACCAGGACTATCCAAAAGTACC 
               
               
                   
                   
                 TGAGGCGAAGGGGTCAATTCAAGCTGTTCGCAACGAGGCGTTT 
               
               
                   
                   
                 TTTATTCCGCTTTACGAACTTTTTTTAACTTATGGTGGGATCTTT 
               
               
                   
                   
                 CGCCTGACATTTGGCCCTAAAAGCTTCCTTATTGTTAGCGATCC 
               
               
                   
                   
                 CTCAATCGCCAAACATATCCTGAAGGATAACGCCAAAGCTTAC 
               
               
                   
                   
                 TCCAAGGGCATTTTAGCGGAAATCTTAGACTTCGTGATGGGTAA 
               
               
                   
                   
                 AGGTCTGATCCCGGCCGATGGCGAGATTTGGCGTCGCCGTCGCC 
               
               
                   
                   
                 GTGCTATCGTGCCGGCTCTTCATCAGAAGTATGTCGCTGCAATG 
               
               
                   
                   
                 ATCAGCCTTTTCGGTGAAGCCTCAGATCGTTTGTGTCAGAAGTT 
               
               
                   
                   
                 AGACGCGGCAGCCTTGAAAGGGGAGGAGGTCGAAATGGAGAG 
               
               
                   
                   
                 CCTGTTCTCACGTTTAACTTTGGACATTATTGGTAAAGCCGTATT 
               
               
                   
                   
                 TAATTATGACTTTGACTCCCTTACCAACGACACGGGGGTGATTG 
               
               
                   
                   
                 AGGCTGTCTACACGGTGCTGCGCGAAGCAGAAGACCGTTCTGT 
               
               
                   
                   
                 TAGCCCTATCCCGGTTTGGGATATCCCTATTTGGAAAGACATCT 
               
               
                   
                   
                 CTCCGCGTCAGCGTAAAGTTGCAACAAGCTTGAAATTAATTAAC 
               
               
                   
                   
                 GACACACTTGATGACTTAATCGCAACATGTAAGCGTATGGTTGA 
               
               
                   
                   
                 GGAAGAAGAGTTACAGTTTCATGAGGAATATATGAACGAACGT 
               
               
                   
                   
                 GATCCGTCCATTTTGCATTTTTTACTTGCTAGTGGTGACGACGTC 
               
               
                   
                   
                 AGCTCCAAACAGTTACGTGACGACCTGATGACTATGCTGATCGC 
               
               
                   
                   
                 TGGTCATGAAACATCAGCAGCTGTGTTGACCTGGACATTTTACT 
               
               
                   
                   
                 TGCTTACAACGGAGCCGTCAGTGGTGGCGAAGCTTCAGGAGGA 
               
               
                   
                   
                 GGTGGACTCCGTTATTGGAGACCGCTTCCCTACAATTCAAGACA 
               
               
                   
                   
                 TGAAGAAATTAAAATATACAACTCGCGTTATGAACGAATCTCT 
               
               
                   
                   
                 GCGCTTGTATCCCCAACCACCTGTTTTAATTCGTCGCTCTATTGA 
               
               
                   
                   
                 CAATGACATCTTAGGCGAATACCCTATCAAGCGTGGTGAGGAC 
               
               
                   
                   
                 ATCTTCATTTCTGTGTGGAATTTGCATCGCAGCCCCTTGCACTG 
               
               
                   
                   
                 GGATGATGCGGAAAAATTTAATCCCGAGCGTTGGCCACTGGAT 
               
               
                   
                   
                 GGTCCCAACCCAAATGAGACGAATCAGAATTTCAGCTATTTGCC 
               
               
                   
                   
                 TTTTGGCGGAGGACCGCGTAAGTGTATTGGAGATATGTTCGCCA 
               
               
                   
                   
                 GCTTTGAGAATGTGGTAGCCATCGCCATGCTGATCCGTCGTTTT 
               
               
                   
                   
                 AACTTTCAAATTGCACCAGGAGCGCCCCCCGTAAAGATGACCA 
               
               
                   
                   
                 CCGGTGCTACTATTCATACGACCGAAGGCTTGAAATTAACAGTA 
               
               
                   
                   
                 ACGAAACGTACAAAACCATTAGATATTCCGTCCGTTCCGATCCT 
               
               
                   
                   
                 TCCTATGGACACTTCGCGCGATGAAGTTAGCTCGGCCCTTTCTT 
               
               
                   
                   
                 AA 
               
               
                   
               
               
                 22 
                 trLUT1 
                 ATGAGTTCCATCGAAAAACCGAAACCGAAACTGGAAACCAACT 
               
               
                   
                   
                 CATCGAAAAGTCAGTCCTGGGTGTCTCCGGATTGGCTGACCACC 
               
               
                   
                   
                 CTGACCCGTACCCTGAGCAGCGGCAAAAACGACGAATCCGGTA 
               
               
                   
                   
                 TTCCGATCGCAAATGCTAAACTGGATGATGTTGCGGATCTGCTG 
               
               
                   
                   
                 GGCGGCGCGCTGTTTCTGCCGCTGTATAAATGGATGAACGAAT 
               
               
                   
                   
                 ATGGTCCGATTTACCGCCTGGCGGCCGGTCCGCGTAATTTTGTC 
               
               
                   
                   
                 ATTGTGAGCGATCCGGCGATCGCCAAACATGTGCTGCGTAACT 
               
               
                   
                   
                 ATCCGAAATACGCCAAAGGCCTGGTGGCAGAAGTTAGCGAATT 
               
               
                   
                   
                 TCTGTTCGGCTCTGGTTTTGCAATTGCTGAAGGTCCGCTGTGGA 
               
               
                   
                   
                 CCGCCCGTCGCCGTGCAGTGGTTCCGAGTCTGCACCGCCGTTAC 
               
               
                   
                   
                 CTGTCCGTTATCGTCGAACGCGTGTTCTGCAAATGTGCGGAACG 
               
               
                   
                   
                 TCTGGTTGAAAAACTGCAACCGTATGCTGAAGATGGCAGTGCG 
               
               
                   
                   
                 GTGAATATGGAAGCCAAATTTTCCCAAATGACCCTGGACGTTAT 
               
               
                   
                   
                 CGGTCTGTCACTGTTCAACTACAACTTCGATTCACTGACCACGG 
               
               
                   
                   
                 ACTCGCCGGTCATCGAAGCAGTGTACACCGCTCTGAAAGAAGC 
               
               
                   
                   
                 GGAACTGCGCTCTACGGATCTGCTGCCGTATTGGAAAATTGACG 
               
               
                   
                   
                 CACTGTGCAAAATCGTTCCGCGCCAGGTCAAAGCAGAAAAAGC 
               
               
                   
                   
                 TGTCACCCTGATTCGTGAAACGGTGGAAGACCTGATTGCGAAA 
               
               
                   
                   
                 TGTAAAGAAATCGTGGAACGCGAAGGCGAACGTATTAACGATG 
               
               
                   
                   
                 AAGAATACGTTAATGATGCGGACCCGAGCATCCTGCGCTTCCTG 
               
               
                   
                   
                 CTGGCCTCTCGTGAAGAAGTCAGTTCCGTGCAGCTGCGCGATGA 
               
               
                   
                   
                 CCTGCTGTCTATGCTGGTCGCCGGCCATGAAACCACGGGTTCAG 
               
               
                   
                   
                 TGCTGACCTGGACGCTGTATCTGCTGTCGAAAAACTCATCGGCC 
               
               
                   
                   
                 CTGCGTAAAGCACAAGAAGAAGTTGATCGCGTCCTGGAAGGTC 
               
               
                   
                   
                 GTAACCCGGCCTTCGAAGACATTAAAGAACTGAAATACATCAC 
               
               
                   
                   
                 CCGCTGCATCAATGAAAGTATGCGTCTGTACCCGCATCCGCCGG 
               
               
                   
                   
                 TTCTGATTCGCCGTGCACAGGTCCCGGATATTCTGCCGGGCAAC 
               
               
                   
                   
                 TATAAAGTGAATACCGGTCAAGACATTATGATCTCCGTTTACAA 
               
               
                   
                   
                 TATCCACCGCAGCTCTGAAGTCTGGGAAAAAGCAGAAGAATTT 
               
               
                   
                   
                 CTGCCGGAACGTTTCGATATTGACGGCGCTATCCCGAACGAAA 
               
               
                   
                   
                 CCAACACGGATTTCAAATTCATCCCGTTTAGCGGCGGTCCGCGC 
               
               
                   
                   
                 AAATGTGTGGGTGATCAGTTCGCTCTGATGGAAGCGATCGTGG 
               
               
                   
                   
                 CGCTGGCCGTGTTTCTGCAACGTCTGAACGTGGAACTGGTTCCG 
               
               
                   
                   
                 GATCAAACCATTAGCATGACCACGGGCGCCACGATCCACACCA 
               
               
                   
                   
                 CGAATGGTCTGTATATGAAAGTTTCTCAACGTTAA 
               
               
                   
               
            
           
         
       
     
     1-3. Analysis of Lutein Production 
     An LUT1 strain was constructed by introducing the pLUT1, pLUT2 and pLUT3 plasmids into the WLGB-RPP strain (Choi, H. S., et al.,  Appl Environ Microb  76, 3097-3105, 2010) constructed by the present inventors in the study of lycopene production. Glycerol known to be superior to glucose in previous carotenoid production studies was used as a carbon source, and expression of foreign genes was induced using 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG). 
     As a result of flask culture, it was confirmed that only alpha-carotene (0.54 mg l −1 ) and beta-carotene (0.61 mg l −1 ) were produced, and lutein was not produced ( FIG. 1 b   ). 
     Strain name: LUT1 
     Genetic status: W3110 (ΔlacI ΔgdhA ΔgpmB ptrc dxs idi ispA pps), crtE, crtB, crtI, trLUT2, trLCYB, trLUT5, trLUT1, and ATR 
     Example 2. Construction of Substrate Tunnel 
     2-1. Selection of Genes for Constructing Substrate Tunnel 
     The reason why lutein was not produced in Example 1 was believed to be because the promiscuous enzyme LCYB reduced the metabolic flux to the lutein pathway by converting lycopene into beta-carotene. Thus, to reduce the metabolic flux to the beta-carotene pathway and to increase the metabolic flux to the alpha-carotene, three substrate tunnels were designed using the CipA scaffold protein. The first substrate tunnel is a substrate tunnel that is based on CipA and links phytoene to alpha-carotene by bringing CrtI, trLUT2 and trLCYB together, the second is a substrate tunnel that links phytoene to delta-carotene (δ-carotene) by bringing CrtI and trLUT2 together, and the third is a substrate tunnel that links lycopene to alpha-carotene by bringing trLUT2 and trLCYB together. 
     It is known that CipA from  Photorhabdus luminescens  produces protein crystalline inclusions (PCIs) with enzymatic activity in  E. coli , and that PCIs are formed in the same way even when CipA is fused with other enzymes (Wang, Y., et al.,  Acs Synth Biol  6, 826-836, 2017). Thus, pLUT4 was constructed by replacing crtI of pLUT1 with cipA-crtI, and pLUT5 was constructed by replacing trLUT2 and trLCYB of pLUT2 with trLUT2 and trLCYB, respectively. In addition, pLUT6 was also constructed by replacing only trLUT2 of pLUT2 with cipA-trLUT2. 
     2-2. Plasmid Construction 
     A detailed plasmid construction procedure is as follows. cipA from  P. luminescens  was synthesized and used after codon optimization. To construct pLUT4, cipA and crtI were amplified using a combination of cipA_F1/cipA_R1 primers and a combination of crtI_F2/crtI_R primers, respectively, and then ligated together by PCR and inserted into the crtI-inserted position of pLUT1 by Gibson&#39;s method. The plasmid was linearized by PCR amplification using crtB_F/crtE_R2 primers. 
     To construct pLUT5, cipA was amplified with cipA_F2/R2 and cipA_F3/R3 primers, and then ligated by PCR to the trLUT2 and trLCYB gene fragments used in the construction of pLUT2. The cipA-trLUT2 and cipA-trLCYB gene fragments were inserted into the SacI/XbaI and XbaI/PstI cleavage sites of the pBBR1Tac plasmid, respectively, thereby constructing pLUT5. pLUT6 was constructed by inserting and amplifying the cipA-trLUT2 and trLCYB gene fragments in the same manner. 
     Table 3 below the sequences of the primers used in construction of the plasmids. 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 SEQ ID 
                   
                   
               
               
                 NO 
                 Name 
                 Sequence 
               
               
                   
               
             
            
               
                 23 
                 cipA_F1 
                 AACTCGCTGCCGTCAGTTAATTCACACAG 
               
               
                   
                   
                 GAAACAATGATTAATGACATGCATCCTT 
               
               
                   
               
               
                 24 
                 cipA_R1 
                 CAATTACCGTAGTTGGTTTCATCATAGAG 
               
               
                   
                   
                 ATTTCTACGCAATTA 
               
               
                   
               
               
                 25 
                 crtI_F2 
                 ATGAAACCAACTACGGTAAT 
               
               
                   
               
               
                 26 
                 crtI_R 
                 TAAATCAGATCCTCCAGCATCA 
               
               
                   
               
               
                 27 
                 crtB_F 
                 TGATGCTGGAGGATCTGATTTAATTCACAC 
               
               
                   
                   
                 AGGAAACAATGAATAATCCGTCGTTACTCA 
               
               
                   
               
               
                 28 
                 crtE_R2 
                 TTAACTGACGGCAGCGAGTT 
               
               
                   
               
               
                 29 
                 cipA_F2 
                 AGACAGGAGCTCTTCACACAGGAAACAATG 
               
               
                   
                   
                 ATTAATGACATGCATCC 
               
               
                   
               
               
                 30 
                 cipA_R2 
                 GCTGCCACCGCCGGATGCCATCATAGAGAT 
               
               
                   
                   
                 TTCTACGCAATTA 
               
               
                   
               
               
                 31 
                 cipA_F3 
                 AGACAGTCTAGATTCACACAGGAAACAATG 
               
               
                   
                   
                 ATTAATGACATGCATCC 
               
               
                   
               
               
                 32 
                 cipA_R3 
                 GCTCCCCGACACGACCATCATAGAGATTTC 
               
               
                   
                   
                 TACGCAATTA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Gene sequences for constructing substrate 
               
               
                 tunnel 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Name 
                 Sequence 
               
               
                   
               
               
                 33 
                 cipA-crt1 
                 ATGATTAATGACATGCATCCTTCTTTAATTAAAGATAAAGATATAG 
               
               
                   
                   
                 TAGATGACGTAATGCTACGTAGCTGTAAAATCATTGCAATGAAAGT 
               
               
                   
                   
                 TATGCCAGACAAAGTTATGCAAGTTATGGTGACTGTATTAATGCAT 
               
               
                   
                   
                 GATGGCGTATGTGAAGAAATGCTTTTGAAATGGAATCTGCTAGACA 
               
               
                   
                   
                 ATAGAGGCATGGCGATTTATAAAGTTCTGATGGAAGCGCTTTGCGC 
               
               
                   
                   
                 TAAGAAAGATGTGAAAATTAGCACCGTAGGAAAAGTAGGTCCTCTC 
               
               
                   
                   
                 GGCTGCGATTACATTAATTGCGTAGAAATCTCTATGATGAAACCAA 
               
               
                   
                   
                 CTACGGTAATTGGTGCAGGCTTCGGTGGCCTGGCACTGGCAATTCG 
               
               
                   
                   
                 TCTACAGGCTGCGGGGATCCCCGTCTTACTGCTTGAACAACGTGAT 
               
               
                   
                   
                 AAACCCGGCGGTCGGGCTTATGTCTACGAGGATCAGGGGTTTACCT 
               
               
                   
                   
                 TTGATGCAGGCCCGACGGTTATCACCGATCCCAGTGCCATTGAAGA 
               
               
                   
                   
                 ACTGTTTGCACTGGCAGGAAAACAGTTAAAAGAGTATGTCGAACTG 
               
               
                   
                   
                 CTGCCGGTTACGCCGTTTTACCGCCTGTGTTGGGAGTCAGGGAAGG 
               
               
                   
                   
                 TCTTTAATTACGATAACGATCAAACCCGGCTCGAAGCGCAGATTCA 
               
               
                   
                   
                 GCAGTTTAATCCCCGCGATGTCGAAGGTTATCGTCAGTTTCTGGACT 
               
               
                   
                   
                 ATTCACGCGCGGTGTTTAAAGAAGGCTATCTGAAGCTCGGTACTGT 
               
               
                   
                   
                 CCCTTTTTTATCGTTCAGAGACATGCTTCGCGCCGCACCTCAACTGG 
               
               
                   
                   
                 CGAAACTGCAGGCATGGAGAAGCGTTTACAGTAAGGTTGCCAGTTA 
               
               
                   
                   
                 CATCGAAGATGAACATCTGCGCCAGGCGTTTTCTTTCCACTCGCTGT 
               
               
                   
                   
                 TGGTGGGCGGCAATCCCTTCGCCACCTCATCCATTTATACGTTGATA 
               
               
                   
                   
                 CACGCGCTGGAACGTGAGTGGGGCGTCTGGTTTCCGCGTGGCGGCA 
               
               
                   
                   
                 CCGGCGCATTAGTTCAGGGGATGATAAAGCTGTTTCAGGATCTGGG 
               
               
                   
                   
                 TGGCGAAGTCGTGTTAAACGCCAGAGTCAGCCATATGGAAACGAC 
               
               
                   
                   
                 AGGAAACAAGATTGAAGCCGTGCATTTAGAGGACGGTCGCAGGTT 
               
               
                   
                   
                 CCTGACGCAAGCCGTCGCGTCAAATGCAGATGTGGTTCATACCTAT 
               
               
                   
                   
                 CGCGACCTGTTAAGCCAGCACCCTGCCGCGGTTAAGCAGTCCAACA 
               
               
                   
                   
                 AACTGCAGACTAAGCGTATGAGTAACTCTCTGTTTGTGCTCTATTTT 
               
               
                   
                   
                 GGTTTGAATCACCATCATGATCAGCTCGCGCATCACACGGTTTGTTT 
               
               
                   
                   
                 CGGCCCGCGTTACCGCGAACTGATTGACGAGATTTTTAATCATGAT 
               
               
                   
                   
                 GGCCTCGCAGAAGACTTCTCACTTTATCTGCACGCGCCCTGTGTCAC 
               
               
                   
                   
                 GGATTCGTCACTGGCGCCTGAAGGTTGCGGCAGTTACTATGTGTTG 
               
               
                   
                   
                 GCGCCGGTGCCGCATTTAGGCACCGCGAACCTCGACTGGACGGTTG 
               
               
                   
                   
                 AGGGGCCAAAACTACGCGACCGTATTTTTGAGTACCTTGAGCAGCA 
               
               
                   
                   
                 TTACATGCCTGGCTTACGGAGTCAGCTGGTCACGCACCAGATGTTT 
               
               
                   
                   
                 ACGCCGTTTGATTTTCGCGACCAGCTTAATGCCTATCAGGGCTCAGC 
               
               
                   
                   
                 CTTTTCTGTGGAGCCCGTTCTTACCCAGAGCGCCTGGTTTCGGCCGC 
               
               
                   
                   
                 ATAACCGCGATAAAACCATTACTAATCTCTACCTGGTCGGCGCAGG 
               
               
                   
                   
                 CACGCATCCCGGCGCAGGCATTCCTGGCGTCATCGGCTCGGCAAAA 
               
               
                   
                   
                 GCGACAGCAGGTTTGATGCTGGAGGATCTGATTTAA 
               
               
                   
               
               
                 34 
                 cipA-trLUT2 
                 ATGATTAATGACATGCATCCTTCTTTAATTAAAGATAAAGATATAG 
               
               
                   
                   
                 TAGATGACGTAATGCTACGTAGCTGTAAAATCATTGCAATGAAAGT 
               
               
                   
                   
                 TATGCCAGACAAAGTTATGCAAGTTATGGTGACTGTATTAATGCAT 
               
               
                   
                   
                 GATGGCGTATGTGAAGAAATGCTTTTGAAATGGAATCTGCTAGACA 
               
               
                   
                   
                 ATAGAGGCATGGCGATTTATAAAGTTCTGATGGAAGCGCTTTGCGC 
               
               
                   
                   
                 TAAGAAAGATGTGAAAATTAGCACCGTAGGAAAAGTAGGTCCTCTC 
               
               
                   
                   
                 GGCTGCGATTACATTAATTGCGTAGAAATCTCTATGATGGCATCCG 
               
               
                   
                   
                 GCGGTGGCAGCTCTGGCAGCGAATCTTGTGTGGCGGTTCGCGAAGA 
               
               
                   
                   
                 CTTTGCCGATGAAGAAGACTTCGTCAAAGCCGGTGGCTCTGAAATC 
               
               
                   
                   
                 CTGTTTGTGCAGATGCAGCAAAACAAAGACATGGATGAACAAAGT 
               
               
                   
                   
                 AAACTGGTTGATAAACTGCCGCCGATTTCCATCGGTGATGGCGCGC 
               
               
                   
                   
                 TGGACCTGGTTGTGATTGGTTGTGGTCCGGCCGGTCTGGCACTGGC 
               
               
                   
                   
                 AGCTGAAAGCGCCAAACTGGGCCTGAAAGTCGGTCTGATCGGCCCG 
               
               
                   
                   
                 GATCTGCCGTTTACCAACAATTATGGTGTGTGGGAAGATGAATTTA 
               
               
                   
                   
                 ATGACCTGGGCCTGCAAAAATGCATTGAACATGTGTGGCGTGAAAC 
               
               
                   
                   
                 GATCGTTTATCTGGATGACGATAAACCGATTACCATTGGTCGTGCG 
               
               
                   
                   
                 TACGGCCGTGTGAGCCGTCGCCTGCTGCACGAAGAACTGCTGCGTC 
               
               
                   
                   
                 GCTGTGTTGAATCAGGTGTCTCGTATCTGAGTTCCAAAGTTGATAGT 
               
               
                   
                   
                 ATTACGGAAGCATCCGATGGTCTGCGTCTGGTCGCTTGTGACGATA 
               
               
                   
                   
                 ACAATGTGATTCCGTGTCGCCTGGCTACCGTGGCGTCAGGTGCCGC 
               
               
                   
                   
                 CTCGGGTAAACTGCTGCAATATGAAGTTGGTGGCCCGCGTGTCTGT 
               
               
                   
                   
                 GTGCAAACCGCGTATGGTGTTGAAGTCGAAGTGGAAAACTCACCGT 
               
               
                   
                   
                 ACGACCCGGATCAGATGGTTTTTATGGATTATCGTGACTACACCAA 
               
               
                   
                   
                 TGAAAAAGTCCGCAGCCTGGAAGCGGAATATCCGACCTTCCTGTAC 
               
               
                   
                   
                 GCCATGCCGATGACGAAATCACGCCTGTTTTTCGAAGAAACCTGTC 
               
               
                   
                   
                 TGGCCTCGAAAGATGTGATGCCGTTTGACCTGCTGAAAACCAAACT 
               
               
                   
                   
                 GATGCTGCGTCTGGATACGCTGGGCATTCGCATCCTGAAAACCTAT 
               
               
                   
                   
                 GAAGAAGAATGGTCATACATTCCGGTTGGTGGCTCGCTGCCGAACA 
               
               
                   
                   
                 CGGAACAGAAAAATCTGGCATTTGGTGCAGCTGCGAGCATGGTCCA 
               
               
                   
                   
                 TCCGGCTACCGGCTATTCTGTTGTCCGTAGTCTGTCCGAAGCCCCGA 
               
               
                   
                   
                 AATACGCAAGTGTTATTGCTGAAATCCTGCGTGAAGAAACCACGAA 
               
               
                   
                   
                 ACAGATTAACAGCAATATCTCTCGCCAAGCGTGGGATACCCTGTGG 
               
               
                   
                   
                 CCGCCAGAACGTAAACGCCAGCGTGCGTTTTTCCTGTTTGGTCTGGC 
               
               
                   
                   
                 CCTGATTGTGCAATTCGATACGGAAGGCATCCGCAGCTTTTTCCGTA 
               
               
                   
                   
                 CCTTTTTCCGCCTGCCGAAATGGATGTGGCAGGGTTTTCTGGGCAGC 
               
               
                   
                   
                 ACCCTGACGTCTGGCGATCTGGTTCTGTTTGCACTGTATATGTTCGT 
               
               
                   
                   
                 CATTAGCCCGAACAATCTGCGCAAAGGTCTGATTAACCACCTGATC 
               
               
                   
                   
                 TCTGATCCGACCGGCGCTACGATGATCAAAACCTACCTGAAAGTGT 
               
               
                   
                   
                 AA 
               
               
                   
               
               
                 35 
                 cipA-trLCYB 
                 ATGATTAATGACATGCATCCTTCTTTAATTAAAGATAAAGATATAG 
               
               
                   
                   
                 TAGATGACGTAATGCTACGTAGCTGTAAAATCATTGCAATGAAAGT 
               
               
                   
                   
                 TATGCCAGACAAAGTTATGCAAGTTATGGTGACTGTATTAATGCAT 
               
               
                   
                   
                 GATGGCGTATGTGAAGAAATGCTTTTGAAATGGAATCTGCTAGACA 
               
               
                   
                   
                 ATAGAGGCATGGCGATTTATAAAGTTCTGATGGAAGCGCTTTGCGC 
               
               
                   
                   
                 TAAGAAAGATGTGAAAATTAGCACCGTAGGAAAAGTAGGTCCTCTC 
               
               
                   
                   
                 GGCTGCGATTACATTAATTGCGTAGAAATCTCTATGATGGTCGTGTC 
               
               
                   
                   
                 GGGGAGCGCGGCCTTGCTGGATTTAGTCCCCGAAACCAAGAAAGA 
               
               
                   
                   
                 GAACCTGGACTTTGAGTTACCCCTTTATGATACGAGTAAATCCCAA 
               
               
                   
                   
                 GTTGTCGATCTGGCAATCGTCGGAGGGGGCCCGGCAGGGTTAGCAG 
               
               
                   
                   
                 TGGCCCAGCAAGTAAGTGAGGCCGGTCTTAGCGTGTGCTCGATTGA 
               
               
                   
                   
                 CCCCAGTCCCAAGTTGATCTGGCCCAACAATTATGGAGTATGGGTC 
               
               
                   
                   
                 GATGAATTTGAAGCTATGGACCTGTTAGACTGCCTGGATACTACTT 
               
               
                   
                   
                 GGAGTGGAGCAGTTGTGTATGTAGATGAAGGAGTTAAAAAAGATC 
               
               
                   
                   
                 TGTCACGTCCTTATGGACGTGTAAACCGCAAACAGTTAAAGTCAAA 
               
               
                   
                   
                 GATGCTGCAAAAATGTATCACGAACGGTGTGAAGTTTCATCAGTCC 
               
               
                   
                   
                 AAAGTGACCAATGTAGTTCACGAGGAGGCAAATTCAACGGTTGTCT 
               
               
                   
                   
                 GTAGCGACGGTGTGAAAATCCAAGCCAGCGTAGTCCTGGATGCGAC 
               
               
                   
                   
                 GGGGTTTTCGCGTTGCCTGGTTCAATACGACAAGCCCTACAACCCA 
               
               
                   
                   
                 GGCTACCAAGTTGCCTACGGAATTGTAGCAGAAGTAGATGGGCACC 
               
               
                   
                   
                 CCTTCGACGTTGACAAAATGGTTTTTATGGACTGGCGTGATAAGCA 
               
               
                   
                   
                 CCTGGATAGTTACCCAGAATTGAAGGAGCGCAATTCAAAAATTCCT 
               
               
                   
                   
                 ACATTCCTTTATGCGATGCCTTTCTCGTCCAATCGCATCTTCTTGGA 
               
               
                   
                   
                 GGAGACCTCCCTGGTAGCTCGCCCGGGTCTTCGCATGGAAGATATC 
               
               
                   
                   
                 CAGGAACGCATGGCCGCTCGCTTAAAGCACTTAGGTATCAACGTGA 
               
               
                   
                   
                 AGCGTATTGAAGAAGATGAACGCTGTGTAATCCCCATGGGTGGGCC 
               
               
                   
                   
                 ACTGCCCGTGTTACCTCAACGTGTGGTAGGTATCGGTGGCACGGCC 
               
               
                   
                   
                 GGCATGGTTCATCCTAGTACCGGTTACATGGTAGCCCGCACACTGG 
               
               
                   
                   
                 CAGCGGCACCTATTGTCGCAAATGCAATCGTTCGTTATTTGGGTTCC 
               
               
                   
                   
                 CCCTCGTCAAACTCCTTACGCGGAGACCAGTTAAGTGCCGAAGTTT 
               
               
                   
                   
                 GGCGCGACCTTTGGCCCATCGAACGTCGTCGCCAACGCGAATTCTT 
               
               
                   
                   
                 CTGTTTTGGGATGGATATCTTATTAAAACTGGACTTAGATGCGACCC 
               
               
                   
                   
                 GCCGCTTCTTCGACGCATTCTTCGATCTTCAGCCACACTACTGGCAC 
               
               
                   
                   
                 GGATTTTTATCATCGCGTTTATTTCTGCCCGAGTTATTGGTATTCGG 
               
               
                   
                   
                 CTTGAGTCTTTTTTCTCATGCGTCGAACACTTCCCGCCTGGAGATCA 
               
               
                   
                   
                 TGACTAAAGGCACCGTACCATTGGCTAAGATGATCAATAACTTAGT 
               
               
                   
                   
                 GCAGGATCGCGACTAA 
               
               
                   
               
            
           
         
       
     
     2-3. Analysis of Lutein Production 
     Six plasmids, including the newly constructed plasmids, were combined and introduced into the WLGB-RPP strain, thereby constructing three new lutein-producing strains, LUT2, LUT3, and LUT4 (Table 5). 
     These strains were cultured under the same conditions as those for LUT1, and as a result, it was confirmed that the largest amount of lutein (0.84 mg l −1 ) was produced in the LUT4 strain, which possesses pLUT1, pLUT3 and pLUT5 and can bring trLUT2 and trLCYB together ( FIG. 1 b   ). 
     Lutein produced in the LUT4 strain was analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis ( FIG. 3 a   ), and intracellular production of PCIs was analyzed by microscopic observation ( FIG. 3 b   ) and SDS-PAGE analysis ( FIG. 3 d   ). However, it was confirmed that alpha-carotene and beta-carotene were still produced in amounts of 6.74 and 4.50 mg l −1 , respectively ( FIG. 1 b   ). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Genes inserted into each strain 
               
            
           
           
               
               
            
               
                 Strain name 
                 Inserted genes 
               
               
                   
               
               
                 LUT2 
                 crtE, crtB, cipA-crtI, cipA-trLUT2, cipA-trLCYB,  
               
               
                   
                 trLUT5, trLUT1, ATR2 
               
               
                 LUT3 
                 crtE, crtB, cipA-crtI, cipA-trLUT2, trLCYB, trLUT5,  
               
               
                   
                 trLUT1, ATR2 
               
               
                 LUT4 
                 crtE, crtB, crtI, cipA-trLUT2, cipA-trLCYB, trLUT5,  
               
               
                   
                 trLUT1, ATR2 
               
               
                   
               
            
           
         
       
     
     Example 3. Increase in Production Ratio of Alpha-Carotene 
     It was confirmed in Example 2 that lutein was successfully produced, but a significant amount of beta-carotene still remained in the cells ( FIG. 1 b   ). As shown in  FIG. 1 a   , trLCYB is involved in both the reaction that synthesizes beta-carotene from lycopene and the reaction that synthesizes alpha-carotene from delta-carotene. The results in Example 2 suggest that the substrate tunnel composed of trLUT2 and trLCYB is not sufficient to block the metabolic flux to the beta-carotene production pathway. 
     Thus, this time, a point mutation (G404E) in trLCYB was induced so that trLCYB could produce a higher rate of alpha-carotene (Li, Z. R. et al.  Plant Cell  21, 1798-1812, 2009). The trLCYBmut gene was prepared by synthesis, and pLUT5M was constructed by replacing cipA-trLCYB of pLUT5 with cipA-trLCYBmut using the same primers and cloning method. 
     As a result of flask culture of the LUT4M strain having pLUT1, pLUT3 and pLUT5M, it was confirmed that alpha-carotene and beta-carotene were produced in amounts of 9.11 and 1.43 mg l −1  ( FIG. 1 b   ). This means that, due to the G404E mutation induced in trLCYB, the ratio of alpha-carotene production to beta-carotene production increased effectively from 1.50 to 6.37. At this time, alpha-carotene production increased from 6.74 to 9.11 mg l −1 , but lutein production was 1.70 mg l −1 , which did not increase proportionally to alpha-carotene production. As a result, it could be seen that the two P450 enzymes that convert alpha-carotene into lutein are limiting factors. 
     Strain Name: LUT4M 
     Inserted genes: crtE, crtB, crtI, cipA-trLUT2, cipA-trLCYBmut, trLUT5, trLUT1, and ATR2 
     
       
         
           
               
            
               
                 SEQ ID NO 36: cipA-trLCYBmut gene sequence 
               
               
                 ATGATTAATGACATGCATCCTTCTTTAATTAAAGATAAAGATATAGTAGA 
               
               
                   
               
               
                 TGACGTAATGCTACGTAGCTGTAAAATCATTGCAATGAAAGTTATGCCAG 
               
               
                   
               
               
                 ACAAAGTTATGCAAGTTATGGTGACTGTATTAATGCATGATGGCGTATGT 
               
               
                   
               
               
                 GAAGAAATGCTTTTGAAATGGAATCTGCTAGACAATAGAGGCATGGCGAT 
               
               
                   
               
               
                 TTATAAAGTTCTGATGGAAGCGCTTTGCGCTAAGAAAGATGTGAAAATTA 
               
               
                   
               
               
                 GCACCGTAGGAAAAGTAGGTCCTCTCGGCTGCGATTACATTAATTGCGTA 
               
               
                   
               
               
                 GAAATCTCTATGATGGTCGTGTCGGGGAGCGCGGCCTTGCTGGATTTAGT 
               
               
                   
               
               
                 CCCCGAAACCAAGAAAGAGAACCTGGACTTTGAGTTACCCCTTTATGATA 
               
               
                   
               
               
                 CGAGTAAATCCCAAGTTGTCGATCTGGCAATCGTCGGAGGGGGCCCGGCA 
               
               
                   
               
               
                 GGGTTAGCAGTGGCCCAGCAAGTAAGTGAGGCCGGTCTTAGCGTGTGCTC 
               
               
                   
               
               
                 GATTGACCCCAGTCCCAAGTTGATCTGGCCCAACAATTATGGAGTATGGG 
               
               
                   
               
               
                 TCGATGAATTTGAAGCTATGGACCTGTTAGACTGCCTGGATACTACTTGG 
               
               
                   
               
               
                 AGTGGAGCAGTTGTGTATGTAGATGAAGGAGTTAAAAAAGATCTGTCACG 
               
               
                   
               
               
                 TCCTTATGGACGTGTAAACCGCAAACAGTTAAAGTCAAAGATGCTGCAAA 
               
               
                   
               
               
                 AATGTATCACGAACGGTGTGAAGTTTCATCAGTCCAAAGTGACCAATGTA 
               
               
                   
               
               
                 GTTCACGAGGAGGCAAATTCAACGGTTGTCTGTAGCGACGGTGTGAAAAT 
               
               
                   
               
               
                 CCAAGCCAGCGTAGTCCTGGATGCGACGGGGTTTTCGCGTTGCCTGGTTC 
               
               
                   
               
               
                 AATACGACAAGCCCTACAACCCAGGCTACCAAGTTGCCTACGGAATTGTA 
               
               
                   
               
               
                 GCAGAAGTAGATGGGCACCCCTTCGACGTTGACAAAATGGTTTTTATGGA 
               
               
                   
               
               
                 CTGGCGTGATAAGCACCTGGATAGTTACCCAGAATTGAAGGAGCGCAATT 
               
               
                   
               
               
                 CAAAAATTCCTACATTCCTTTATGCGATGCCTTTCTCGTCCAATCGCATC 
               
               
                   
               
               
                 TTCTTGGAGGAGACCTCCCTGGTAGCTCGCCCGGGTCTTCGCATGGAAGA 
               
               
                   
               
               
                 TATCCAGGAACGCATGGCCGCTCGCTTAAAGCACTTAGGTATCAACGTGA 
               
               
                   
               
               
                 AGCGTATTGAAGAAGATGAACGCTGTGTAATCCCCATGGGTGGGCCACTG 
               
               
                   
               
               
                 CCCGTGTTACCTCAACGTGTGGTAGGTATCGGTGGCACGGCCGGCATGGT 
               
               
                   
               
               
                 TCATCCTAGTACCGGTTACATGGTAGCCCGCACACTGGCAGCGGCACCTA 
               
               
                   
               
               
                 TTGTCGCAAATGCAATCGTTCGTTATTTGGGTTCCCCCTCGTCAAACTCC 
               
               
                   
               
               
                 TTACGCGGAGACCAGTTAAGTGCCGAAGTTTGGCGCGACCTTTGGCCCAT 
               
               
                   
               
               
                 CGAACGTCGTCGCCAACGCGAATTCTTCTGTTTTGGGATGGATATCTTAT 
               
               
                   
               
               
                 TAAAACTGGACTTAGATGCGACCCGCCGCTTCTTCGACGCATTCTTCGAT 
               
               
                   
               
               
                 CTTCAGCCACACTACTGGCACGAATTTTTATCATCGCGTTTATTTCTGCC 
               
               
                   
               
               
                 CGAGTTATTGGTATTCGGCTTGAGTCTTTTTTCTCATGCGTCGAACACTT 
               
               
                   
               
               
                 CCCGCCTGGAGATCATGACTAAAGGCACCGTACCATTGGCTAAGATGATC 
               
               
                   
               
               
                 AATAACTTAGTGCAGGATCGCGACTAA 
               
               
                   
               
               
                 SEQ ID NO 58: trLCYBmut gene sequence 
               
               
                 ATGGTCGTGTCGGGGAGCGCGGCCTTGCTGGATTTAGTCCCCGAAACCAA 
               
               
                   
               
               
                 GAAAGAGAACCTGGACTTTGAGTTACCCCTTTATGATACGAGTAAATCCC 
               
               
                   
               
               
                 AAGTTGTCGATCTGGCAATCGTCGGAGGGGGCCCGGCAGGGTTAGCAGTG 
               
               
                   
               
               
                 GCCCAGCAAGTAAGTGAGGCCGGTCTTAGCGTGTGCTCGATTGACCCCAG 
               
               
                   
               
               
                 TCCCAAGTTGATCTGGCCCAACAATTATGGAGTATGGGTCGATGAATTTG 
               
               
                   
               
               
                 AAGCTATGGACCTGTTAGACTGCCTGGATACTACTTGGAGTGGAGCAGTT 
               
               
                   
               
               
                 GTGTATGTAGATGAAGGAGTTAAAAAAGATCTGTCACGTCCTTATGGACG 
               
               
                   
               
               
                 TGTAAACCGCAAACAGTTAAAGTCAAAGATGCTGCAAAAATGTATCACGA 
               
               
                   
               
               
                 ACGGTGTGAAGTTTCATCAGTCCAAAGTGACCAATGTAGTTCACGAGGAG 
               
               
                   
               
               
                 GCAAATTCAACGGTTGTCTGTAGCGACGGTGTGAAAATCCAAGCCAGCGT 
               
               
                   
               
               
                 AGTCCTGGATGCGACGGGGTTTTCGCGTTGCCTGGTTCAATACGACAAGC 
               
               
                   
               
               
                 CCTACAACCCAGGCTACCAAGTTGCCTACGGAATTGTAGCAGAAGTAGAT 
               
               
                   
               
               
                 GGGCACCCCTTCGACGTTGACAAAATGGTTTTTATGGACTGGCGTGATAA 
               
               
                   
               
               
                 GCACCTGGATAGTTACCCAGAATTGAAGGAGCGCAATTCAAAAATTCCTA 
               
               
                   
               
               
                 CATTCCTTTATGCGATGCCTTTCTCGTCCAATCGCATCTTCTTGGAGGAG 
               
               
                   
               
               
                 ACCTCCCTGGTAGCTCGCCCGGGTCTTCGCATGGAAGATATCCAGGAACG 
               
               
                   
               
               
                 CATGGCCGCTCGCTTAAAGCACTTAGGTATCAACGTGAAGCGTATTGAAG 
               
               
                   
               
               
                 AAGATGAACGCTGTGTAATCCCCATGGGTGGGCCACTGCCCGTGTTACCT 
               
               
                   
               
               
                 CAACGTGTGGTAGGTATCGGTGGCACGGCCGGCATGGTTCATCCTAGTAC 
               
               
                   
               
               
                 CGGTTACATGGTAGCCCGCACACTGGCAGCGGCACCTATTGTCGCAAATG 
               
               
                   
               
               
                 CAATCGTTCGTTATTTGGGTTCCCCCTCGTCAAACTCCTTACGCGGAGAC 
               
               
                   
               
               
                 CAGTTAAGTGCCGAAGTTTGGCGCGACCTTTGGCCCATCGAACGTCGTCG 
               
               
                   
               
               
                 CCAACGCGAATTCTTCTGTTTTGGGATGGATATCTTATTAAAACTGGACT 
               
               
                   
               
               
                 TAGATGCGACCCGCCGCTTCTTCGACGCATTCTTCGATCTTCAGCCACAC 
               
               
                   
               
               
                 TACTGGCACGAATTTTTATCATCGCGTTTATTTCTGCCCGAGTTATTGGT 
               
               
                   
               
               
                 ATTCGGCTTGAGTCTTTTTTCTCATGCGTCGAACACTTCCCGCCTGGAGA 
               
               
                   
               
               
                 TCATGACTAAAGGCACCGTACCATTGGCTAAGATGATCAATAACTTAGTG 
               
               
                   
               
               
                 CAGGATCGCGACTAA 
               
            
           
         
       
     
     Example 4. Construction of Electron Tunnel 
     4-1. Selection of Genes for Constructing Electron Tunnel 
     The eukaryotic P450 system generally consists of P450 and P450 reductases associated with membranes. P450 reductase contains flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) factors. Since electrons generated by oxidation of nicotinamide adenine phosphorylated dinucleotide (NADPH) must be transferred to P450 through P450 reductase, the physical distance between the two enzymes is an important factor in electron transfer efficiency. 
     As a result of SDS-PAGE analysis, it was confirmed that the two P450 enzymes were mostly located in the cytoplasm, whereas ATR2 was located in the cell membrane ( FIG. 3 c   ). 
     Thus, with reference to the study wherein bacterial P450 was assembled with ferredoxin and ferredoxin reductase using proliferative cell nuclear antigen (PCNA) constituting a heterogeneous triad (Haslinger, K. et al., Microb) Cell Fact 19, 2020), the present inventors adopted an approach in which electron transfer occurs through an assembly of trLUT5, trLUT1 and ATR2. It was expected that, by doing so, electrons generated in the NADPH oxidation reaction by ATR2 could be immediately transferred to trLUT5 and trLUT1. In the present invention, the CipB scaffold protein from  P. luminescens , which has a smaller molecular size than PCNA and forms stable PCIs separated from CipA, was used as a mediator ( FIG. 1 c   ). pLUT7 was constructed by replacing ATR2, trLUT5 and trLUT1 of pLUT3 with cipB-ATR2, cipB-trLUT5 and cipB-trLUT1, respectively. 
     4-2. Plasmid Construction 
     A detailed method for construction of pLUT7 is as follows. First, cipB from  P. luminescens  was synthesized and used. cipB was amplified with each of a combination of cipB_F1/cipB_R1 primers, a combination of cipB_F1/cipB_R2 primers and a combination of cipB_F1/cipB_R3 primers, and then ligated by PCR to ATR2, trLUT5 and trLUT1 amplified with ATR2_F1/R1, trLUT5_F/R1, and trLUT1_F/R1 primers, respectively. The ligated gene fragments were inserted into the EcoRI/BamHI cleavage sites of the pTrc99a plasmid, thereby constructing pTrc-cipB-ATR2, pTrc-cipB-trLUT5, pTrc-cipB-trLUT1 plasmids, respectively. 
     Thereafter, the cipB-trLUT5 and cipB-trLUT1 gene fragments ligated with the trc promoter were amplified from the pTrc-cipB-trLUT5 and pTrc-cipB-trLUT1 plasmids, respectively, using a combination of pTrc_F1/trLUT5_R2 primers and a combination of pTrc_F2/trLUT1_R2 primers, and then inserted into the BamHI/XbaI and XbaI/SalI cleavage sites of the pTrc-cipB-ATR2 plasmid, respectively. 
     Table 6 below shows the sequences of the primers used in construction of the plasmids. 
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 SEQ ID 
                   
                   
               
               
                 NO 
                 Name 
                 Sequence 
               
               
                   
               
             
            
               
                 37 
                 cipB_F1 
                 AGACAGGAATTCTTCACACAGGAAAC 
               
               
                   
                   
                 AATGATTATCAAGAAAGATATTCTGC 
               
               
                   
               
               
                 38 
                 cipB_R1 
                 GAAGAAGAAGAAGAAGACATGATTTC 
               
               
                   
                   
                 CACACCCACAAT 
               
               
                   
               
               
                 39 
                 cipB_R2 
                 ACTTGAGCTGCTGAACATGATTTCCA 
               
               
                   
                   
                 CACCCACAAT 
               
               
                   
               
               
                 40 
                 cipB_R3 
                 GGTTTTTCGATGGAACTCATGATTTC 
               
               
                   
                   
                 CACACCCACAAT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Gene sequences for constructing electron 
               
               
                 tunnel 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO 
                 Name 
                 Sequence 
               
               
                   
               
               
                 41 
                 cipB-ATR2 
                 ATGATTATCAAGAAAGATATTCTGCTTAACGAAGAACTTATTGT 
               
               
                   
                   
                 TGATGACGACTTGAAGGTGGGTAAAGTAGAGAAAGTCAATATT 
               
               
                   
                   
                 GATATTTTGTCTCCCTCATCGGTTATTGTGTCTTTGAACATCCTT 
               
               
                   
                   
                 GGTGTGGTGGATGACTTCCATCTGCTTTTGGTAGATGATAAGGA 
               
               
                   
                   
                 TAAAGACAAAATCGTATTGTTGTATCTGTCTCTGCTTCGCGTCC 
               
               
                   
                   
                 TTCACGAGAAACTGGACGTCAAGGTTAAGGTCGCGAAATCCAA 
               
               
                   
                   
                 ACTTACAAAGATTAAATACATTGTGGGTGTGGAAATCATGTCTT 
               
               
                   
                   
                 CTTCTTCTTCTTCTTCTACCTCTATGATCGACCTGATGGCTGCTA 
               
               
                   
                   
                 TCATCAAAGGTGAACCGGTTATCGTTTCTGACCCGGCTAACGCT 
               
               
                   
                   
                 TCTGCTTACGAATCTGTTGCTGCTGAACTGTCTTCTATGTTAATT 
               
               
                   
                   
                 GAGAATCGTCAGTTTGCTATGATCGTTACAACATCCATCGCGGT 
               
               
                   
                   
                 CCTTATTGGTTGTATCGTTATGTTGGTCTGGCGCCGCTCTGGTTC 
               
               
                   
                   
                 CGGTAACTCTAAACGTGTGGAACCGCTTAAACCGCTGGTGATC 
               
               
                   
                   
                 AAACCTCGTGAGGAGGAAATCGACGATGGACGTAAAAAAGTAA 
               
               
                   
                   
                 CAATCTTTTTCGGAACGCAGACTGGCACTGCGGAAGGTTTTGCC 
               
               
                   
                   
                 AAGGCATTAGGTGAGGAAGCTAAAGCTCGTTATGAAAAGACGC 
               
               
                   
                   
                 GCTTCAAGATTGTTGATCTGGACGATTACGCTGCAGACGATGAT 
               
               
                   
                   
                 GAATACGAGGAAAAATTAAAAAAAGAGGATGTAGCTTTCTTCT 
               
               
                   
                   
                 TCTTAGCTACGTATGGCGATGGTGAACCGACAGATAATGCCGCT 
               
               
                   
                   
                 CGTTTTTATAAGTGGTTTACCGAAGGCAATGATCGTGGTGAGTG 
               
               
                   
                   
                 GTTGAAAAACTTAAAATATGGGGTTTTCGGGCTGGGCAATCGTC 
               
               
                   
                   
                 AATACGAGCACTTTAACAAGGTCGCGAAAGTGGTCGATGACAT 
               
               
                   
                   
                 TCTGGTTGAGCAAGGCGCACAGCGTCTGGTACAAGTAGGGTTA 
               
               
                   
                   
                 GGGGATGATGACCAGTGTATCGAAGATGATTTCACAGCTTGGC 
               
               
                   
                   
                 GCGAAGCATTGTGGCCCGAGTTGGATACGATTCTGCGCGAAGA 
               
               
                   
                   
                 GGGCGATACGGCTGTTGCCACACCCTACACAGCCGCAGTATTA 
               
               
                   
                   
                 GAGTATCGCGTAAGCATCCATGATAGCGAGGATGCCAAATTCA 
               
               
                   
                   
                 ATGATATTAACCTTGCTAACGGAAACGGGTATACAGTTTTTGAC 
               
               
                   
                   
                 GCTCAACATCCGTATAAGGCCAACGTTGCGGTCAAACGTGAAT 
               
               
                   
                   
                 TGCACACCCCGGAGTCCGACCGTTCCTGTATCCATCTGGAATTT 
               
               
                   
                   
                 GATATTGCGGGATCAGGTTTAACATACGAAACTGGAGATCACG 
               
               
                   
                   
                 TTGGTGTTCTGTGCGATAACTTATCCGAGACGGTGGATGAGGCA 
               
               
                   
                   
                 CTGCGCCTTTTAGACATGTCCCCTGACACGTATTTTAGCTTGCAT 
               
               
                   
                   
                 GCTGAAAAAGAGGACGGTACTCCGATCAGTAGCTCGCTGCCAC 
               
               
                   
                   
                 CGCCGTTTCCACCGTGCAATTTACGCACGGCTTTAACACGTTAC 
               
               
                   
                   
                 GCGTGCCTGTTGTCATCTCCTAAGAAATCCGCCTTAGTGGCTTT 
               
               
                   
                   
                 GGCTGCACACGCTAGTGATCCCACTGAGGCCGAGCGCTTGAAA 
               
               
                   
                   
                 CACTTAGCAAGCCCTGCAGGTAAAGACGAGTACTCCAAGTGGG 
               
               
                   
                   
                 TAGTAGAGTCACAGCGTAGTTTATTGGAGGTGATGGCCGAGTTT 
               
               
                   
                   
                 CCTAGTGCGAAGCCACCGTTGGGAGTTTTCTTTGCCGGGGTGGC 
               
               
                   
                   
                 TCCGCGTTTGCAACCACGTTTTTATAGCATCAGTAGTTCTCCAA 
               
               
                   
                   
                 AAATCGCCGAGACTCGCATTCACGTTACATGTGCCCTGGTCTAC 
               
               
                   
                   
                 GAAAAAATGCCGACTGGGCGCATCCACAAGGGTGTATGCTCGA 
               
               
                   
                   
                 CTTGGATGAAGAACGCCGTACCCTACGAAAAGTCTGAAAACTG 
               
               
                   
                   
                 CAGCTCGGCGCCAATCTTCGTACGCCAGTCCAATTTCAAGTTGC 
               
               
                   
                   
                 CGTCAGATTCAAAGGTACCGATCATTATGATCGGTCCAGGAAC 
               
               
                   
                   
                 GGGGTTAGCTCCGTTCCGTGGGTTCTTACAGGAACGCTTAGCAC 
               
               
                   
                   
                 TGGTCGAGTCGGGGGTAGAATTGGGCCCCTCCGTCTTGTTTTTC 
               
               
                   
                   
                 GGGTGTCGTAACCGTCGCATGGACTTCATCTATGAAGAAGAGC 
               
               
                   
                   
                 TGCAACGTTTCGTGGAAAGTGGGGCGCTTGCTGAACTGTCGGTG 
               
               
                   
                   
                 GCGTTTTCCCGCGAAGGACCCACGAAAGAATATGTTCAACACA 
               
               
                   
                   
                 AAATGATGGACAAAGCGTCGGATATCTGGAACATGATTTCACA 
               
               
                   
                   
                 GGGCGCTTATTTATATGTATGTGGCGATGCGAAAGGCATGGCG 
               
               
                   
                   
                 CGTGACGTCCACCGTTCTCTGCACACCATTGCGCAAGAGCAAG 
               
               
                   
                   
                 GTAGCATGGATTCAACGAAAGCAGAAGGCTTCGTGAAGAATTT 
               
               
                   
                   
                 ACAAACCTCTGGGCGCTATCTTCGTGATGTGTGGTAA 
               
               
                   
               
               
                 42 
                 cipB-trLUT5 
                 ATGATTATCAAGAAAGATATTCTGCTTAACGAAGAACTTATTGT 
               
               
                   
                   
                 TGATGACGACTTGAAGGTGGGTAAAGTAGAGAAAGTCAATATT 
               
               
                   
                   
                 GATATTTTGTCTCCCTCATCGGTTATTGTGTCTTTGAACATCCTT 
               
               
                   
                   
                 GGTGTGGTGGATGACTTCCATCTGCTTTTGGTAGATGATAAGGA 
               
               
                   
                   
                 TAAAGACAAAATCGTATTGTTGTATCTGTCTCTGCTTCGCGTCC 
               
               
                   
                   
                 TTCACGAGAAACTGGACGTCAAGGTTAAGGTCGCGAAATCCAA 
               
               
                   
                   
                 ACTTACAAAGATTAAATACATTGTGGGTGTGGAAATCATGTTCA 
               
               
                   
                   
                 GCAGCTCAAGTAACGGCCGCGATCCACTTGAGGAAAACTCAGT 
               
               
                   
                   
                 ACCGAATGGCGTGAAAAGCCTGGAGAAATTGCAGGAGGAAAA 
               
               
                   
                   
                 ACGCCGTGCTGAATTGTCGGCCCGTATCGCATCCGGTGCATTCA 
               
               
                   
                   
                 CCGTTCGTAAGAGCAGCTTCCCCAGCACTGTCAAGAATGGTCTT 
               
               
                   
                   
                 AGCAAAATCGGGATTCCTAGCAACGTATTGGATTTTATGTTCGA 
               
               
                   
                   
                 CTGGACAGGAAGTGACCAGGACTATCCAAAAGTACCTGAGGCG 
               
               
                   
                   
                 AAGGGGTCAATTCAAGCTGTTCGCAACGAGGCGTTTTTTATTCC 
               
               
                   
                   
                 GCTTTACGAACTTTTTTTAACTTATGGTGGGATCTTTCGCCTGAC 
               
               
                   
                   
                 ATTTGGCCCTAAAAGCTTCCTTATTGTTAGCGATCCCTCAATCG 
               
               
                   
                   
                 CCAAACATATCCTGAAGGATAACGCCAAAGCTTACTCCAAGGG 
               
               
                   
                   
                 CATTTTAGCGGAAATCTTAGACTTCGTGATGGGTAAAGGTCTGA 
               
               
                   
                   
                 TCCCGGCCGATGGCGAGATTTGGCGTCGCCGTCGCCGTGCTATC 
               
               
                   
                   
                 GTGCCGGCTCTTCATCAGAAGTATGTCGCTGCAATGATCAGCCT 
               
               
                   
                   
                 TTTCGGTGAAGCCTCAGATCGTTTGTGTCAGAAGTTAGACGCGG 
               
               
                   
                   
                 CAGCCTTGAAAGGGGAGGAGGTCGAAATGGAGAGCCTGTTCTC 
               
               
                   
                   
                 ACGTTTAACTTTGGACATTATTGGTAAAGCCGTATTTAATTATG 
               
               
                   
                   
                 ACTTTGACTCCCTTACCAACGACACGGGGGTGATTGAGGCTGTC 
               
               
                   
                   
                 TACACGGTGCTGCGCGAAGCAGAAGACCGTTCTGTTAGCCCTAT 
               
               
                   
                   
                 CCCGGTTTGGGATATCCCTATTTGGAAAGACATCTCTCCGCGTC 
               
               
                   
                   
                 AGCGTAAAGTTGCAACAAGCTTGAAATTAATTAACGACACACT 
               
               
                   
                   
                 TGATGACTTAATCGCAACATGTAAGCGTATGGTTGAGGAAGAA 
               
               
                   
                   
                 GAGTTACAGTTTCATGAGGAATATATGAACGAACGTGATCCGT 
               
               
                   
                   
                 CCATTTTGCATTTTTTACTTGCTAGTGGTGACGACGTCAGCTCCA 
               
               
                   
                   
                 AACAGTTACGTGACGACCTGATGACTATGCTGATCGCTGGTCAT 
               
               
                   
                   
                 GAAACATCAGCAGCTGTGTTGACCTGGACATTTTACTTGCTTAC 
               
               
                   
                   
                 AACGGAGCCGTCAGTGGTGGCGAAGCTTCAGGAGGAGGTGGAC 
               
               
                   
                   
                 TCCGTTATTGGAGACCGCTTCCCTACAATTCAAGACATGAAGAA 
               
               
                   
                   
                 ATTAAAATATACAACTCGCGTTATGAACGAATCTCTGCGCTTGT 
               
               
                   
                   
                 ATCCCCAACCACCTGTTTTAATTCGTCGCTCTATTGACAATGAC 
               
               
                   
                   
                 ATCTTAGGCGAATACCCTATCAAGCGTGGTGAGGACATCTTCAT 
               
               
                   
                   
                 TTCTGTGTGGAATTTGCATCGCAGCCCCTTGCACTGGGATGATG 
               
               
                   
                   
                 CGGAAAAATTTAATCCCGAGCGTTGGCCACTGGATGGTCCCAA 
               
               
                   
                   
                 CCCAAATGAGACGAATCAGAATTTCAGCTATTTGCCTTTTGGCG 
               
               
                   
                   
                 GAGGACCGCGTAAGTGTATTGGAGATATGTTCGCCAGCTTTGA 
               
               
                   
                   
                 GAATGTGGTAGCCATCGCCATGCTGATCCGTCGTTTTAACTTTC 
               
               
                   
                   
                 AAATTGCACCAGGAGCGCCCCCCGTAAAGATGACCACCGGTGC 
               
               
                   
                   
                 TACTATTCATACGACCGAAGGCTTGAAATTAACAGTAACGAAA 
               
               
                   
                   
                 CGTACAAAACCATTAGATATTCCGTCCGTTCCGATCCTTCCTAT 
               
               
                   
                   
                 GGACACTTCGCGCGATGAAGTTAGCTCGGCCCTTTCTTAA 
               
               
                   
               
               
                 43 
                 cipB-trLUT1 
                 ATGATTATCAAGAAAGATATTCTGCTTAACGAAGAACTTATTGT 
               
               
                   
                   
                 TGATGACGACTTGAAGGTGGGTAAAGTAGAGAAAGTCAATATT 
               
               
                   
                   
                 GATATTTTGTCTCCCTCATCGGTTATTGTGTCTTTGAACATCCTT 
               
               
                   
                   
                 GGTGTGGTGGATGACTTCCATCTGCTTTTGGTAGATGATAAGGA 
               
               
                   
                   
                 TAAAGACAAAATCGTATTGTTGTATCTGTCTCTGCTTCGCGTCC 
               
               
                   
                   
                 TTCACGAGAAACTGGACGTCAAGGTTAAGGTCGCGAAATCCAA 
               
               
                   
                   
                 ACTTACAAAGATTAAATACATTGTGGGTGTGGAAATCATGAGTT 
               
               
                   
                   
                 CCATCGAAAAACCGAAACCGAAACTGGAAACCAACTCATCGAA 
               
               
                   
                   
                 AAGTCAGTCCTGGGTGTCTCCGGATTGGCTGACCACCCTGACCC 
               
               
                   
                   
                 GTACCCTGAGCAGCGGCAAAAACGACGAATCCGGTATTCCGAT 
               
               
                   
                   
                 CGCAAATGCTAAACTGGATGATGTTGCGGATCTGCTGGGCGGC 
               
               
                   
                   
                 GCGCTGTTTCTGCCGCTGTATAAATGGATGAACGAATATGGTCC 
               
               
                   
                   
                 GATTTACCGCCTGGCGGCCGGTCCGCGTAATTTTGTCATTGTGA 
               
               
                   
                   
                 GCGATCCGGCGATCGCCAAACATGTGCTGCGTAACTATCCGAA 
               
               
                   
                   
                 ATACGCCAAAGGCCTGGTGGCAGAAGTTAGCGAATTTCTGTTC 
               
               
                   
                   
                 GGCTCTGGTTTTGCAATTGCTGAAGGTCCGCTGTGGACCGCCCG 
               
               
                   
                   
                 TCGCCGTGCAGTGGTTCCGAGTCTGCACCGCCGTTACCTGTCCG 
               
               
                   
                   
                 TTATCGTCGAACGCGTGTTCTGCAAATGTGCGGAACGTCTGGTT 
               
               
                   
                   
                 GAAAAACTGCAACCGTATGCTGAAGATGGCAGTGCGGTGAATA 
               
               
                   
                   
                 TGGAAGCCAAATTTTCCCAAATGACCCTGGACGTTATCGGTCTG 
               
               
                   
                   
                 TCACTGTTCAACTACAACTTCGATTCACTGACCACGGACTCGCC 
               
               
                   
                   
                 GGTCATCGAAGCAGTGTACACCGCTCTGAAAGAAGCGGAACTG 
               
               
                   
                   
                 CGCTCTACGGATCTGCTGCCGTATTGGAAAATTGACGCACTGTG 
               
               
                   
                   
                 CAAAATCGTTCCGCGCCAGGTCAAAGCAGAAAAAGCTGTCACC 
               
               
                   
                   
                 CTGATTCGTGAAACGGTGGAAGACCTGATTGCGAAATGTAAAG 
               
               
                   
                   
                 AAATCGTGGAACGCGAAGGCGAACGTATTAACGATGAAGAATA 
               
               
                   
                   
                 CGTTAATGATGCGGACCCGAGCATCCTGCGCTTCCTGCTGGCCT 
               
               
                   
                   
                 CTCGTGAAGAAGTCAGTTCCGTGCAGCTGCGCGATGACCTGCTG 
               
               
                   
                   
                 TCTATGCTGGTCGCCGGCCATGAAACCACGGGTTCAGTGCTGAC 
               
               
                   
                   
                 CTGGACGCTGTATCTGCTGTCGAAAAACTCATCGGCCCTGCGTA 
               
               
                   
                   
                 AAGCACAAGAAGAAGTTGATCGCGTCCTGGAAGGTCGTAACCC 
               
               
                   
                   
                 GGCCTTCGAAGACATTAAAGAACTGAAATACATCACCCGCTGC 
               
               
                   
                   
                 ATCAATGAAAGTATGCGTCTGTACCCGCATCCGCCGGTTCTGAT 
               
               
                   
                   
                 TCGCCGTGCACAGGTCCCGGATATTCTGCCGGGCAACTATAAA 
               
               
                   
                   
                 GTGAATACCGGTCAAGACATTATGATCTCCGTTTACAATATCCA 
               
               
                   
                   
                 CCGCAGCTCTGAAGTCTGGGAAAAAGCAGAAGAATTTCTGCCG 
               
               
                   
                   
                 GAACGTTTCGATATTGACGGCGCTATCCCGAACGAAACCAACA 
               
               
                   
                   
                 CGGATTTCAAATTCATCCCGTTTAGCGGCGGTCCGCGCAAATGT 
               
               
                   
                   
                 GTGGGTGATCAGTTCGCTCTGATGGAAGCGATCGTGGCGCTGG 
               
               
                   
                   
                 CCGTGTTTCTGCAACGTCTGAACGTGGAACTGGTTCCGGATCAA 
               
               
                   
                   
                 ACCATTAGCATGACCACGGGCGCCACGATCCACACCACGAATG 
               
               
                   
                   
                 GTCTGTATATGAAAGTTTCTCAACGTTAA 
               
               
                   
               
            
           
         
       
     
     4-3. Analysis of Lutein Production 
     As a result of flask culture of the LUT5M strain having pLUT1, pLUT5M and pLUT7, it was confirmed that lutein was produced in the LUT5M strain in an amount of (5.80 mg l −1 ), which was 3.41 times more than that produced in the LUT4M strain ( FIG. 1 d   ). The intracellular production of PCIs composed of trLUT5, trLUT1 and ATR2 was analyzed by microscopic observation and SDS-PAGE analysis ( FIG. 3 e   ). As a result, it was confirmed that alpha-carotene and beta-carotene were produced together in amounts of 8.23 and 1.34 mg l −1 , respectively. 
     Strain name: LUT5M 
     Inserted genes: crtE, crtB, crtI, cipA-trLUT2, cipA-trLCYBmut, cipB-trLUT5, cipB-trLUT1, and cipB-ATR2 
     Example 5. Enhancement of C5 Heme Production Pathway 
     5-1. Selection of Genes for Enhancing C5 Heme Production Pathway 
     In order to further increase lutein production, the activities of trLUT5 and trLUT1 to convert residual alpha-carotene into lutein should be increased. Two P450 enzymes use heme, which serves as a mediator to transfer electrons from NADPH to oxygen, as a cofactor. If the intracellular heme concentration is low, the function of P450 may be reduced ( FIG. 2 a   ). Thus, to enhance the metabolic flux of the C5 heme biosynthesis pathway (Zhao, X. R., et al.,  Nat Catal  1, 720-728, 2018),  E. coli  hemA fbr , hemL, hemB and hemH genes (which encode feedback-resistant glutamyl-tRNA reductase, glutamate-1-semialdehyde 2,1-aminomutase, porphobilinogen synthase, and ferrochelatase, respectively) were overexpressed. 
     pHEM2, which expresses hemAfbr and hemL, and pHEM2, which expresses hemA fbr , hemL, hemB and hemH genes, were constructed and then introduced into LUT5M, thereby constructing LUT5MH1 and LUT5MH2 strains, respectively. 
     5-2. Plasmid Construction 
     A detailed plasmid construction method is as follows. The hemA fbr , hemL, hemB and hemH genes were all amplified from the genome of  E. coli  W3110. First, to construct pHEM2, hemA fbr  and hemL genes were amplified using hemA_F1/R primers and hemL_F/R1 primers, respectively, and then inserted into the NcoI/BamHI and BamHI/PstI cleavage sites of the pTrcCDF plasmid, respectively. 
     Before construction of pHEM2, the pTrcCDF plasmid linearized with pTrcCDF_F/R, hemB amplified with hemB_F/R, and hemH amplified with hemH_F/R1 were assembled together by Gibson&#39;s method, thereby constructing a pTrc-hemBH plasmid. A hemBH operon ligated with the trc promoter was amplified from pTrc-hemBH using a combination of pTrc_F3/hemH_R2 primers, and then inserted into the SphI cleavage site of pHEM1, thereby constructing pHEM2. 
     Table 8 below shows the sequences of the plasmids used in plasmid construction. 
     
       
         
           
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO 
                 Name 
                 Sequence 
               
               
                   
               
             
            
               
                 44 
                 hemA_F1 
                 AGACAGCCATGGGTACCAAGAAGCTTTTAGCA 
               
               
                   
                   
                 CTCGGTATCAAC 
               
               
                   
               
               
                 45 
                 hemA_R 
                 AGACAGGGATCCCTACTCCAGCCCGAGGCT 
               
               
                   
               
               
                 46 
                 hemL_F 
                 AGACAGGGATCCTTTCACACAGGAAACAGACC 
               
               
                   
                   
                 ATGAGTAAGTCTGAAAATCTTTAC 
               
               
                   
               
               
                 47 
                 hemL_R1 
                 AGACAGCTGCAGTCACAACTTCGCAAACAC 
               
               
                   
               
               
                 48 
                 pTrcCDF_F 
                 TCTAGAGTCGACCTGCAG 
               
               
                   
               
               
                 49 
                 pTrcCDF_R 
                 TCTGTTTCCTGTGTGAAATT 
               
               
                   
               
               
                 50 
                 hemB_F 
                 AACAATTTCACACAGGAAACAATGACAGACTT 
               
               
                   
                   
                 AATCCAACG 
               
               
                   
               
               
                 51 
                 hemB_R 
                 TTAACGCAGAATCTTCTTCTC 
               
               
                   
               
               
                 52 
                 hemH_F 
                 CTGAGAAGAAGATTCTGCGTTAATTCACACAG 
               
               
                   
                   
                 GAAACAATGCGTCAGACTAAAACC 
               
               
                   
               
               
                 53 
                 hemH_R1 
                 CTGCAGGTCGACTCTAGATTAGCGATACGCGG 
               
               
                   
                   
                 CAAC 
               
               
                   
               
               
                 54 
                 pTrc_F3 
                 AGACAGGCATGCTTGACAATTAATCATCCGGC 
               
               
                   
               
               
                 55 
                 hemH_R2 
                 AGACAGGCATGCTTAGCGATACGCGGCAAC 
               
               
                   
               
            
           
         
       
     
     5-3. Analysis of Lutein Production 
     As a result of flask culture, it was confirmed that the LUT5MH1 and LUT5MH2 strains produced lutein in amounts of 10.34 and 6.04 mg l −1 , respectively ( FIG. 2 b   ). This means that the increase in heme production had a positive effect on the increase in the synthesis of lutein from alpha-carotene catalyzed by the two P450 enzymes. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Genes inserted into each strain 
               
            
           
           
               
               
            
               
                 Strain name 
                 Inserted genes 
               
               
                   
               
               
                 LUT5MH1 
                 crtE, crtB, crtI, cipA-trLUT2, cipA-trLCYBmut,  
               
               
                   
                 cipB-trLUT5, cipB-trLUT1, cipB-ATR2, hemA fbr ,  
               
               
                   
                 hemL 
               
               
                 LUT5MH2 
                 crtE, crtB, crtI, cipA-trLUT2, cipA-trLCYBmut,  
               
               
                   
                 cipB-trLUT5, cipB-trLUT1, cipB-ATR2,  
               
               
                   
                 hem fbr , hemL, hemB, hemH 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                 SEQ ID NO 56: hemA fbr  gene 
               
               
                 ATGGGTACCAAGAAGCTTTTAGCACTCGGTATCAACCATAAAACGGCACC 
               
               
                   
               
               
                 TGTATCGCTGCGAGAACGTGTATCGTTTTCGCCGGATAAGCTCGATCAGG 
               
               
                   
               
               
                 CGCTTGACAGCCTGCTTGCGCAGCCGATGGTGCAGGGCGGCGTGGTGCTG 
               
               
                   
               
               
                 TCGACGTGCAACCGCACGGAACTTTATCTTAGCGTTGAAGAGCAGGACAA 
               
               
                   
               
               
                 CCTGCAAGAGGCGTTAATCCGCTGGCTTTGCGATTATCACAATCTTAATG 
               
               
                   
               
               
                 AAGAAGATCTGCGTAAAAGCCTCTACTGGCATCAGGATAACGACGCGGTT 
               
               
                   
               
               
                 AGCCATTTAATGCGTGTTGCCAGCGGCCTGGATTCACTGGTTCTGGGGGA 
               
               
                   
               
               
                 GCCGCAGATCCTCGGTCAGGTTAAAAAAGCGTTTGCCGATTCGCAAAAAG 
               
               
                   
               
               
                 GTCATATGAAGGCCAGCGAACTGGAACGCATGTTCCAGAAATCTTTCTCT 
               
               
                   
               
               
                 GTCGCGAAACGCGTTCGCACTGAAACAGATATCGGTGCCAGCGCTGTGTC 
               
               
                   
               
               
                 TGTCGCTTTTGCGGCTTGTACGCTGGCGCGGCAGATCTTTGAATCGCTCT 
               
               
                   
               
               
                 CTACGGTCACAGTGTTGCTGGTAGGCGCGGGCGAAACTATCGAGCTGGTG 
               
               
                   
               
               
                 GCGCGTCATCTGCGCGAACACAAAGTACAGAAGATGATTATCGCCAACCG 
               
               
                   
               
               
                 CACTCGCGAACGTGCCCAAATTCTGGCAGATGAAGTCGGCGCGGAAGTGA 
               
               
                   
               
               
                 TTGCCCTGAGTGATATCGACGAACGTCTGCGCGAAGCCGATATCATCATC 
               
               
                   
               
               
                 AGTTCCACCGCCAGCCCGTTACCGATTATCGGGAAAGGCATGGTGGAGCG 
               
               
                   
               
               
                 CGCATTAAAAAGCCGTCGCAACCAACCAATGCTGTTGGTGGATATTGCCG 
               
               
                   
               
               
                 TTCCGCGCGATGTTGAGCCGGAAGTTGGCAAACTGGCGAATGCTTATCTT 
               
               
                   
               
               
                 TATAGCGTTGATGATCTGCAAAGCATCATTTCGCACAACCTGGCGCAGCG 
               
               
                   
               
               
                 TAAAGCCGCAGCGGTTGAGGCGGAAACTATTGTCGCTCAGGAAACCAGCG 
               
               
                   
               
               
                 AATTTATGGCGTGGCTGCGAGCACAAAGCGCCAGCGAAACCATTCGCGAG 
               
               
                   
               
               
                 TATCGCAGCCAGGCAGAGCAAGTTCGCGATGAGTTAACCGCCAAAGCGTT 
               
               
                   
               
               
                 AGCGGCCCTTGAGCAGGGCGGCGACGCGCAAGCCATTATGCAGGATCTGG 
               
               
                   
               
               
                 CATGGAAACTGACTAACCGCTTGATCCATGCGCCAACGAAATCACTTCAA 
               
               
                   
               
               
                 CAGGCCGCCCGTGACGGGGATAACGAACGCCTGAATATTCTGCGCGACAG 
               
               
                   
               
               
                 CCTCGGGCTGGAGTAG 
               
               
                   
               
               
                 SEQ ID NO 57: hemL gene 
               
               
                 ATGAGTAAGTCTGAAAATCTTTACAGCGCAGCGCGCGAGCTGATCCCTGG 
               
               
                   
               
               
                 CGGTGTGAACTCCCCTGTTCGCGCCTTTACTGGCGTGGGCGGCACTCCAC 
               
               
                   
               
               
                 TGTTTATCGAAAAAGCGGACGGCGCTTATCTGTACGATGTTGATGGCAAA 
               
               
                   
               
               
                 GCCTATATCGATTATGTCGGTTCCTGGGGGCCGATGGTGCTGGGCCATAA 
               
               
                   
               
               
                 CCATCCGGCAATCCGCAATGCCGTGATTGAAGCCGCCGAGCGTGGTTTAA 
               
               
                   
               
               
                 GCTTTGGTGCACCAACCGAAATGGAAGTGAAAATGGCGCAACTGGTGACC 
               
               
                   
               
               
                 GAACTGGTCCCGACCATGGATATGGTGCGCATGGTGAACTCCGGCACTGA 
               
               
                   
               
               
                 AGCGACCATGAGCGCCATCCGCCTGGCCCGTGGTTTTACCGGTCGCGACA 
               
               
                   
               
               
                 AAATTATTAAATTTGAAGGGTGTTACCATGGTCACGCTGACTGCCTGCTG 
               
               
                   
               
               
                 GTGAAAGCCGGTTCTGGCGCACTCACGTTAGGCCAGCCAAACTCGCCGGG 
               
               
                   
               
               
                 CGTTCCGGCAGATTTCGCCAAATATACCTTAACCTGTACTTATAATGATC 
               
               
                   
               
               
                 TGGCTTCTGTACGCGCCGCATTTGAGCAATACCCGCAAGAGATTGCCTGT 
               
               
                   
               
               
                 ATTATCGTCGAGCCGGTGGCAGGCAATATGAACTGTGTTCCGCCGCTGCC 
               
               
                   
               
               
                 AGAGTTCCTGCCAGGTCTGCGCGCGCTGTGCGACGAATTTGGCGCGTTGC 
               
               
                   
               
               
                 TGATCATCGATGAAGTGATGACCGGTTTCCGCGTAGCGCTAGCTGGCGCA 
               
               
                   
               
               
                 CAGGATTATTACGGCGTAGTGCCAGATTTAACCTGCCTCGGCAAAATCAT 
               
               
                   
               
               
                 CGGCGGTGGAATGCCGGTAGGCGCATTCGGTGGTCGTCGTGATGTAATGG 
               
               
                   
               
               
                 ATGCGCTGGCCCCGACGGGTCCGGTCTATCAGGCGGGTACGCTTTCCGGT 
               
               
                   
               
               
                 AACCCGATTGCGATGGCAGCGGGTTTCGCCTGTCTGAATGAAGTCGCGCA 
               
               
                   
               
               
                 GCCGGGCGTTCACGAAACGCTGGATGAGCTGACAACACGTCTGGCAGAAG 
               
               
                   
               
               
                 GTCTGCTGGAAGCGGCAGAAGAAGCCGGAATTCCGCTGGTCGTTAACCAC 
               
               
                   
               
               
                 GTTGGCGGCATGTTCGGTATTTTCTTTACCGACGCCGAGTCCGTGACGTG 
               
               
                   
               
               
                 CTATCAGGATGTGATGGCCTGTGACGTGGAACGCTTTAAGCGTTTCTTCC 
               
               
                   
               
               
                 ATATGATGCTGGACGAAGGTGTTTACCTGGCACCGTCAGCGTTTGAAGCG 
               
               
                   
               
               
                 GGCTTTATGTCCGTGGCGCACAGCATGGAAGATATCAATAACACCATCGA 
               
               
                   
               
               
                 TGCTGCACGTCGGGTGTTTGCGAAGTTGTGA 
               
            
           
         
       
     
     Example 6. Culture Condition Optimization 
     Next, culture conditions having effects on the production of exogenous enzymes including P450 and the growth of cells were optimized. 
     Culture media (MR and R/2), culture temperatures (22, 25, 28 and 30° C.), and IPTG concentrations (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 mM) for inducing gene expression were tested under various conditions. As a result, it was confirmed that lutein production was higher when R/2 medium was used than when MR medium was used ( FIG. 2 c   ). Thus, the remaining culture conditions were tested using R/2 medium. 
     R/2 medium (pH 6.8) contains, per liter, 2 g (NH 4 ) 2 HPO 4 , 6.75 g KH 2 PO 4 , 0.85 g citric acid, 0.7 g MgSO 4 .7H 2 O, 5 ml trace metal solution (TMS) [containing, per liter of 5 M HCl solution, 10 g FeSO 4 .7H 2 O, 2.25 g ZnSO 4 .7H 2 O, 1 g CuSO 4 .5H 2 O, 0.5 g MnSO 4 .5H 2 O, 0.23 g Na 2 B 4 O 7 .10H 2 O, 2 g CaCl 2 .2H 2 O, and 0.1 g (NH 4 ) 6 Mo 7 O 24 ], and MR medium (pH 6.8) contains, per liter, 4 g (NH 4 ) 2 HPO 4 , 6.67 g KH 2 PO 4 , 0.8 g citric acid, 0.8 g MgSO 4 .7H 2 O, and 5 ml TMS. 
     Each of the media was further supplemented with 20 g l −1  of glycerol, 3 g l −1  of yeast extract, 50 mg l −1  of kanamycin (Km) , 34 mg l −1  of chloramphenicol (Cm) , 100 mg l −1  of ampicillin (Ap), and 50 mg l −1  of spectinomycin (Spc). 
     As a result, it was confirmed that, when the culture temperature was lowered to 28° C., lutein production increased to 16.29 mg l −1  ( FIG. 2 d   ), and when the IPTG concentration was reduced to 0.05 mM, lutein production further increased to 23.50 mg l −1  ( FIG. 2 e   ). Under these conditions, it was confirmed that alpha-carotene production and beta-carotene production decreased to 3.04 and 0.66 mg l −1 , respectively. 
     Next, fed-batch culture of the LUT5MH1 strain was performed in R/2 medium. After the initially supplied carbon source was completely exhausted, a feed solution containing 800 g l −1  of glycerol was supplied using a pH-stat nutrient supply strategy. With reference to the flask culture results described above, the culture temperature was maintained at 28° C. from seed culture. When the optical density at 600 nm (OD600) reached 20 to 30, IPTG was added to a final concentration of 0.05 mM. 
     It was confirmed that, when fed-batch culture was performed under these conditions, 25.47 mg l −1  (0.44 mg gDCW −1 ) of lutein was produced with a productivity of 0.64 mg l −1  h −1  ( FIG. 4 a   ). This value was similar to the concentration of lutein produced by flask culture. Optimal culture conditions at the flask level may differ from the optimal culture conditions for fed-batch culture having different conditions including the final cell density level. 
     Thus, optimization of culture conditions at the fed-batch level was performed. First, the induction phase was shortened by increasing the culture temperature before addition of IPTG from 28° C. to 37° C. ( FIG. 4 b   ). Considering the high cell density, the IPTG concentration was reduced to 0.2 mM ( FIG. 4 c   ), 0.5 mM ( FIGS. 4 d   ), and 1 mM ( FIG. 4 e   ), and then lutein production was compared. 
     As a result, it was confirmed that, when fed batch culture was performed under the conditions where the initial cell culture temperature was increased to 37° C. and the culture temperature was reduced to 28° C. when the OD 600  reached 23.4, followed by addition of 0.5 mM of IPTG, the lutein production concentration and productivity increased the most to 133.44 mg l −1  (2.17 mg gDCW −1 ) and 2.72 mg l −1  h −1 , respectively ( FIG. 4 d   ).  FIG. 2 f    shows photographs of the incubator during fed-batch culture of the LUT5MH1 strain. 
     Example 7. Increase in Lutein Production through Carbon Starvation During Fed-Batch Culture 
     It was confirmed that, when the supply solution was not added for 2 hours after the depletion of the initially supplied carbon source during the fed-batch culture, lutein production and productivity further increased to 194.20 mg l −1  (3.38 mg gDCW −1 ) and 3.35 mg l −1  h −1 , respectively ( FIG. 4 f   ). 
     To confirm the consistency of these results, fed-batch culture was performed again under the same conditions, and as a result, it was confirmed that lutein production and productivity further increased to 218.0 mg l −1  (4.01 mg gDCW −1 ) and 5.01 mg l −1  h −1 , respectively ( FIG. 2 g   ). In order to find out the factors causing the increase in lutein production, the transcriptomes of cells cultured under carbon-starved conditions and cells cultured under existing conditions were analyzed and compared. As a result it was confirmed that the change in the expression levels of stress response genes increased the final concentration of lutein by slowing the oxidation rate of intracellular carotenoids. 
     Although the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto. 
     INDUSTRIAL APPLICABILITY 
     Using the highly efficient lutein-producing recombinant microbial strain according to the present invention, it is possible to replace an existing lutein production method that relies on labor-intensive and inefficient plant extraction and to produce lutein in a more environmentally friendly and sustainable way. In addition, the strain development strategy used in the present invention is useful because it may be used to construct a recombinant strain for the efficient production of useful compounds with complex metabolic pathways and to establish an efficient production method, and it may be applied throughout the gradually expanding biochemical market.