WO2025013841A1 - Carotenoid having asymmetric skeleton and carotenoid having fluorescent asymmetric skeleton - Google Patents

Abstract

[Problem] The present invention addresses the problem of providing a carotenoid having a fluorescent asymmetric skeleton. [Solution] By reacting this carotenoid which has an asymmetric skeleton having a chain X and a chain Y having different numbers of carbon atoms with an enzyme that desaturates single bonds adjacent to three conjugated double bond structures present in a linking moiety between the chain X and the chain Y, a carotenoid which has a chain X and a chain Y and has five conjugated double bond structures is produced. The produced carotenoid has an asymmetric skeleton and has fluorescence property.

Description

Carotenoids with asymmetric skeletons and fluorescent carotenoids with asymmetric skeletons

The present invention relates to carotenoids having an asymmetric skeleton, carotenoids having a fluorescent asymmetric skeleton, a method for producing carotenoids having a fluorescent asymmetric skeleton, a method for screening mutant diapophytoene synthase, and a method for screening enzymes involved in the carotenoid precursor supply pathway.

Carotenoids are natural pigments that are widely distributed in nature, and are of great industrial value as pigments and color enhancers that produce yellow to red colors. Carotenoids have a large molecular weight and are highly hydrophobic, so they tend to remain in cell membranes and have a strong tendency to accumulate in cells and tissues. In other words, carotenoids have the advantage of being highly retained in tissues as pigments (Non-Patent Document 1). However, as a rule, carotenoids are not fluorescent.

Phytofluene is known as an exceptionally fluorescent carotenoid (Non-Patent Document 2, Non-Patent Document 3).
However, commercially available methods for synthesizing fluorescent carotenoids have not been established.

Annu. Rev. Microbiol., 51,629-659 (1997) Photobiol., 74 549-557 (2001) Chemical Physics Letter, 58, (1989) Microbiol. Mol. Biol. Rev., 69, 51-78 (2005)

Carotenoid pigments are synthesized by desaturation of phytoene compounds. Phytofluene, which is exceptionally fluorescent, has a polyene-type chromophore with five conjugated double bonds, which is obtained by one-step desaturation of the CC bond adjacent to the chromophore of the phytoene precursor (which has three conjugated double bonds). 2-, 3-, 4-, and 5-step desaturases are known in nature, but no one-step enzymes are known (Non-Patent Document 4). The greatest reason for the difficulty in the biosynthesis of phytofluene-type pigments is that carotenoid biosynthesis begins with phytoene, a precursor with perfect 2-fold symmetry. If a desaturase can desaturate one end of the carotenoid skeleton (positions 11-12), it will also act on the opposite end (positions 11'-12'). It is very difficult to create a pathway in which a desaturase acts only on one end of the carotenoid skeleton to produce a one-step product as the final product. Since both ends of the carotenoid skeleton have identical local structures, desaturases recognize both ends, resulting in symmetrical two-step desaturation products (e.g., γ-carotene has seven conjugated double bonds).
The present invention aims to provide a carotenoid having a fluorescent asymmetric skeleton, which has an X chain and a Y chain and five conjugated double bond structures. The present invention also provides a carotenoid having an asymmetric skeleton, which has asymmetric X chain and Y chain and three conjugated double bond structures at the connection between the X chain and the Y chain. Here, preferred examples of the carotenoid having an asymmetric skeleton provided by the present invention are carotenoids having a skeleton smaller than that of natural carotenoids, such as C25 and C20. Furthermore, the present invention also provides a method for producing a carotenoid having a fluorescent asymmetric skeleton, a method for screening a mutant diapophytoene synthase, and a method for screening an enzyme related to a carotenoid precursor supply pathway.

The present inventors conceived a synthetic route capable of synthesizing carotenoids having an asymmetric skeleton as a method for selectively and efficiently producing one-step desaturation products. The present inventors first produced a carotenoid having asymmetric X and Y chains and three conjugated double bond structures at the junction between the X and Y chains (a carotenoid having an asymmetric skeleton) by bimolecular condensation of two different sizes of prenyl diphosphates (a prenyl diphosphate having n isoprenoid units and a prenyl diphosphate having m isoprenoid units). The present inventors then reacted the carotenoid having an asymmetric skeleton with an enzyme that desaturates the single bonds adjacent to the three conjugated double bond structures at the junction between the X and Y chains, thereby producing a carotenoid having X and Y chains and five conjugated double bond structures, and confirmed that the carotenoid was a carotenoid having an asymmetric skeleton with fluorescent properties. The present inventors further developed a method for screening mutant diapophytoene synthases and a method for screening enzymes involved in the carotenoid precursor supply pathway, using a carotenoid having a fluorescent asymmetric skeleton and the carotenoid synthesis pathway.
Based on the above, the present inventors have completed the present invention.
In the above description, "adjacent single bonds" in "a carotenoid having an asymmetric skeleton, a single bond adjacent to three conjugated double bond structures present at the connecting portion between X and Y chains..." refers to "single bonds at adjacent α and β positions" with respect to the three conjugated double bond structures present at the connecting portion between X and Y chains, and "the closest single bond that can conjugate with the triene structure present at the connecting portion between X and Y chains." In this specification, when "a single bond adjacent to three conjugated double bond structures present at the connecting portion between X and Y chains is an adjacent single bond," all "adjacent single bonds" have the same meaning.

In other words, in some aspects, the present invention provides the following 1. to 42.

1. A carotenoid represented by any one of the following formulas:
2. A carotenoid represented by the following formula:
3. A carotenoid represented by the following formula:
4. A carotenoid represented by the following formula:
5. A carotenoid represented by the following formula:
6. A carotenoid represented by the following formula:
7. A carotenoid represented by the following formula:
8. A carotenoid represented by the following formula:
9. A carotenoid represented by the following formula:
10. A carotenoid represented by the following formula:
11. A carotenoid represented by the following formula:

12. A method for producing a carotenoid having an X chain and a Y chain and having five conjugated double bond structures, comprising the steps of:
A)
1) in a carotenoid having an X chain and a Y chain and having three conjugated double bond structures at the junction between the X chain and the Y chain, a step of desaturating a single bond adjacent to the three conjugated double bond structures at the junction between the X chain and the Y chain;
Or,
B)
1) A step of producing a carotenoid having an X chain and a Y chain and having three conjugated double bond structures at the connecting portion between the X chain and the Y chain by condensing two molecules of a prenyl diphosphate having n isoprenoid units and a prenyl diphosphate having m isoprenoid units;
2) a step of desaturating a single bond adjacent to three conjugated double bonds present at the connection between the X chain and the Y chain of the carotenoid produced in 1);
Here, X is a structure in which n isoprenoid units (C5) are linearly bonded, Y is a structure in which m isoprenoid units (C5) are linearly bonded, and the carbon at the end of the X chain forms a double bond with the carbon at the end of the Y chain to form three conjugated double bond structures, and n and m are integers of 1 to 8, but are different from each other.
A manufacturing method comprising:
13. The method according to item 12 above, wherein the desaturation step is a step of reacting with an enzyme that desaturates a single bond adjacent to three conjugated double bonds present at the link between the X chain and the Y chain.
14. The method according to item 13 above, wherein the enzyme is an enzyme that substantially desaturates only the single bond on the X chain side adjacent to the three conjugated double bond structures.
15. The method according to item 12 above, wherein the X chain has 15 carbon atoms, the Y chain has 10 carbon atoms, and only the single bond in the X chain adjacent to the three conjugated double bond structures is substantially unsaturated.
16. The method according to item 12 above, wherein the X chain has 15 carbon atoms, the Y chain has 5 carbon atoms, and only the single bond in the X chain adjacent to the three conjugated double bond structures is substantially unsaturated.
17. The method according to item 12 above, wherein the X chain has 20 carbon atoms, the Y chain has 25 carbon atoms, and only the single bond in the X chain adjacent to the three conjugated double bond structures is substantially unsaturated.
18. The method according to item 12 above, wherein the X chain has 20 carbon atoms, the Y chain has 30 carbon atoms, and only the single bonds in the X chain adjacent to the three conjugated double bond structures are substantially unsaturated.
19. The method according to item 12 above, wherein the X chain has 15 carbon atoms, the Y chain has 30 carbon atoms, and only the single bonds in the X chain adjacent to the three conjugated double bond structures are substantially unsaturated.
20. The method according to item 12 above, wherein the X chain has 20 carbon atoms, the Y chain has 15 carbon atoms, and only the single bond in the X chain adjacent to the three conjugated double bond structures is substantially unsaturated.
21. The method according to item 12 above, wherein the X chain has 15 carbon atoms, the Y chain has 25 carbon atoms, and only the single bond in the X chain adjacent to the three conjugated double bond structures is substantially unsaturated.
22. The method according to any one of items 12 to 21 above, wherein the carotenoid having an X chain and a Y chain and having five conjugated double bond structures is a fluorescent carotenoid.

23. The production method is carried out in a cell into which the following genes (1) to (4) have been introduced or which resides therein:
(1) a prenyl diphosphate synthesis gene having n isoprenoid units; (2) a prenyl diphosphate synthesis gene having m isoprenoid units; (3) an enzyme gene that condenses two molecules of a prenyl diphosphate having n isoprenoid units and a prenyl diphosphate having m isoprenoid units; (4) an enzyme gene that desaturates a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain;
and,
13. The method according to item 12 above, characterized in that the production method is carried out by culturing the cells in a medium containing an organic solvent while extracting the carotenoid having an X chain and a Y chain and having five conjugated double bonds from the solvent.
24. The production method according to the preceding item 23, wherein the enzyme that desaturates a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain is an enzyme selected from CrtI, CrtN _L155Q , CrtI _{E69K, R152C, F419L} , and PDS.
25. The method according to the preceding item 23 or 24, wherein the enzyme that bicondenses a prenyl diphosphate having n isoprenoid units and a prenyl diphosphate having m isoprenoid units is a diapophytoene synthase (CrtM) into which a mutation has been introduced, and the mutation includes at least L160W, or F26A and W38A.

26. A method for screening for mutant diapophytoene synthases having the activity of bicondensing two different sizes of prenyl diphosphates, comprising the steps of: introducing a diapophytoene synthase (CrtM) gene library into cells capable of synthesizing a carotenoid having an X chain and a Y chain and five conjugated double bond structures, from a carotenoid having an X chain and a Y chain and three conjugated double bond structures; and selecting the cells using the fluorescence intensity of the cells as an index.
27. The method for screening a mutant diapophytoene synthase having an activity of condensing two prenyl diphosphate molecules of different sizes according to the preceding item 26, wherein the X chain has 15 carbon atoms, the Y chain has 10 carbon atoms, and single bonds adjacent to three conjugated double bond structures in the X chain are desaturated.
28. The screening method according to the preceding item 26 or 27, wherein the cell capable of synthesizing a carotenoid having an X chain and a Y chain and having five conjugated double bond structures from the carotenoid having an X chain and a Y chain and having three conjugated double bond structures is a cell into which an enzyme gene for desaturating a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain has been introduced or which contains the gene internally.
29. The screening method according to the preceding item 28, wherein the enzyme that desaturates a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain is an enzyme selected from CrtI, CrtN _L155Q , CrtI _{E69K,R152C,F419L} , and PDS.

30. A mutant diapophytoene synthase (CrtM) having an activity of condensing two molecules of prenyl diphosphate with different sizes, in which one or more mutations selected from L160W and A245T are introduced into a wild type.
31. The mutant diapophytoene synthase (CrtM) according to the above item 30, wherein the mutations L160W, E180G and A245T are introduced into the wild-type.
32. The mutant diapophytoene synthase (CrtM) according to the above item 30 or 31, further comprising any one of G138A, G138S, G138C, and G138T mutations introduced thereinto.
33. The mutant diapophytoene synthase (CrtM) according to the above item 30 or 31, further comprising a G138A or G138S mutation introduced therein.
34. The mutant diapophytoene synthase (CrtM) according to the above item 30 or 31, further comprising a G138A mutation introduced therein.
35. A method for synthesizing 20-carbon phytoene having a C5+C15 structure and/or 25-carbon phytoene having a C10+C15 structure, comprising culturing a transformed cell expressing a mutant diapophytoene synthase (CrtM) having a triple mutation of L160W, E180G and A245T.
36. A method for synthesizing 20-carbon phytoene having a C5+C15 structure and/or 25-carbon phytoene having a C10+C15 structure, comprising culturing a transformed cell expressing a mutant diapophytoene synthase (CrtM) having a quadruple mutation of G138A, L160W, E180G and A245T.
37. The method according to the above item 35 or 36, wherein the transformed cell expresses a mutant IPK, IPK3m (V73I, Y41V, K204G), together with the mutant CrtM.
38. The method according to 37 above, wherein the culture is carried out in a medium containing 1 to 10 mM DMAOH.
39. A method for screening an enzyme involved in a carotenoid precursor supply pathway, comprising the following steps (1) and (2):
(1) introducing a gene library in which mutations have been introduced into genes encoding enzymes involved in the carotenoid precursor supply pathway (herein, a gene library in which mutations have been introduced refers to a gene library in which deletions, substitutions, insertions and/or additions have been introduced into the base sequence of a wild-type gene) into a cell capable of synthesizing a carotenoid having an X chain and a Y chain and having five conjugated double bond structures from a carotenoid having an X chain and a Y chain and having three conjugated double bond structures;
(2) culturing the transfected cells, and then selecting cells in which the function of an enzyme involved in the carotenoid precursor supply pathway is improved from the cultured cell population using the fluorescence intensity of the cells as an indicator.
40. The screening method according to the preceding paragraph 39, wherein the cell capable of synthesizing a carotenoid having an X chain and a Y chain and five conjugated double bond structures from a carotenoid having an X chain and a Y chain and having three conjugated double bond structures is a cell into which an enzyme gene having an activity of synthesizing a carotenoid having an X chain and a Y chain and having three conjugated double bond structures by condensing two molecules of prenyl diphosphate of different sizes has been introduced or is a cell into which an enzyme gene for desaturating a single bond adjacent to the three conjugated double bond structures present at the junction between the X chain and the Y chain ....
41. The screening method according to the preceding item 40, wherein the X chain has 15 carbon atoms, the Y chain has 10 carbon atoms, and single bonds adjacent to three conjugated double bond structures in the X chain are desaturated.
42. The screening method according to any one of the preceding paragraphs 39 to 41, wherein the enzyme involved in the carotenoid precursor supply pathway is DXS.

The present invention can provide the following:
1) Carotenoids with asymmetric skeletons;
2) Carotenoids with fluorescent asymmetric skeletons;
3) A method for producing a carotenoid having a fluorescent asymmetric skeleton;
4) A method for screening for mutant diapophytoene synthase; and 5) a method for screening for enzymes involved in the carotenoid precursor supply pathway.

Structural example 1 of a "carotenoid having three conjugated double bond structures" and a "carotenoid having five conjugated double bond structures" is shown. C35-phytofluene synthesis in E. coli production system (cell color). (a) Plasmid expression/construct diagram. (b) Colony. Results of C35-phytofluene synthesis in an E. coli production system (spectroscopic characteristics of the extracted components). (a) Absorption spectrum of the extract, (b) Fluorescence (emission, excitation) spectrum of the compound. Results of C35-phytofluene synthesis in an E. coli production system (HPLC) ((a)-(c)), and visible absorption spectrum of the pigments (d). (a) C35 backbone plasmid only, (b) C35 backbone plasmid + PDS, (c) C40 backbone plasmid + PDS (Plac-fdsY81M-Plac-crtMF26A,W38A, pACYC ori, Cm marker), (d) Visible absorption spectra of the major pigment of the C35 backbone (C35(5)) and the major pigment of the C40 backbone (C40(7)). Production of C50-phytofluene in E. coli. (a) Plasmid expression/construct diagram. (b) Fluorescence spectrum. (c) Absorption spectrum. Production of C45-phytofluene (C25+20-phytofluene) in E. coli. (a) Plasmid expression/construct diagram. (b) Fluorescence spectrum. (c) Absorption spectrum. Production of C45-phytofluene (C30+15-phytofluene) in E. coli. (a) Plasmid expression/construct diagram. (b) Fluorescence spectrum. (c) Absorption spectrum. Production of C25-phytofluene in E. coli. (a) Plasmid expression/construct diagram. (b) Fluorescence spectrum. (c) Absorption spectrum. Sequential extraction of C25 phytofluene with organic solvents. (a) Plasmid expression/construct diagram. (b) HPLC analysis of the lipid-soluble fraction in the cell pellet. (c) HPLC analysis of the HPLC fraction of the dodecane phase layered on top of the culture medium. Peak1; C30-neurosporene, Peak2; C30-lycopene, Peak3; C25-ζ-carotene, Peak4; C25-neurosporene. (a) Plasmid expression/construct diagram. (b) Colony diagram of the CrtM library. Carotenoid synthesis profile of the CrtM mutant obtained by fluorescence screening. (a) Fluorescence intensity in the dodecane layer. (b) Total carotenoid synthesis amount in the dodecane layer. (c) Total carotenoid synthesis amount in the cell pellet. Results of functional assessment of CrtM mutants. Mutations introduced into second generation CrtM mutants. FIG. 1 shows the construct of FDS(T121A)-CrtM(F26S/W38A) (Example 1). FIG. 1 shows the construct of pBAD-PDS (Example 1). Construct diagram of HexPS-fds(Y81M)-CrtM(22/26/38/180/233) (Example 2). Construct diagram of pBAD-CrtI(E69K, R152C, F419L) (Examples 2 and 3). Construct diagram of HepST(107L)-fds(81M)-CrtM(26/38/241) (Example 3). Construct diagram of HexPS-fds-CrtM(22/38/180) (Example 4). Construct diagram of pBAD-CrtN(L155Q) (Example 4). Construct diagram of gps-CrtM(L160W) (Example 5). Construct diagram of dxsmutC-IspHmut12 (Example 5). Diagram of CrtM library construct (Example 7). Construct diagram of ispA(S80F)-pBAD/CrtI (Example 7). Production of C40-phytofluene in Escherichia coli (a) Plasmid expression/construct diagram (b) Fluorescence spectrum (c) Absorption spectrum (Example 8) E. coli production of C20-phytoene by oversupplying DMAPP (a) Plasmid expression/construct diagram and biosynthetic pathway, (b) HPLC of accumulated carotenoids with and without added DMAOH, (c) Comparison of production amounts of accumulated C20 (top of graph), C25 (middle of graph), and C30 carotenoids (bottom of graph) with and without added DMAOH (Example 9) Construct diagram of pJ211-IPKmut (Example 9). FIG. 1 shows the construct of pUC-CrtM (Example 9). FACS analysis results of the Dxs mutant library using the C20+15-phytofluene synthetic strain (Example 10). The horizontal axis represents fluorescence intensity, and the vertical axis represents the number of bacteria showing the fluorescence intensity on the horizontal axis. The further to the right the histogram is shifted, the stronger the fluorescence emitted by the bacterial cell group (fluorescence excitation laser wavelength: 375 nm, emission wavelength: 500 nm). Top: E. coli strain expressing only the C20+15-phytoene synthetic pathway (pAC-plac-fdsT121A-plac-crtMF26A,W38A+pBAD-PDS). Middle: E. coli strain additionally expressing Dxswt in addition to the C20+15-phytofluene synthetic strain (pAC-plac-fdsT121A-plac-crtMF26A,W38A + pUC-pBAD-pds+pLS-pT5-dxswt). Because Dxs is the main bottleneck enzyme, simply expressing wt shifted the median of the group to the right. Bottom: E. coli strain in which the Dxs library was introduced into the C20+15-phytofluene synthesis strain (pAC-plac-fdsT121A-plac-crtMF26A,W38A + pUC-pBAD-pds+pLS-pT5-dxslib). Synthetic route to the fluorescent asymmetric scaffold carotenoids of the present invention. Structural example 2 of a "carotenoid having three conjugated double bond structures" and a "carotenoid having five conjugated double bond structures" is shown. Specific growth rate of IPK3m/CrtW co-expressing cells in medium supplemented with DMAOH (shown as relative growth rate, with the growth rate at 0 mM DMAOH taken as 1) (Example 11). Phytoene synthesis amount per cell of the crtM gene-introduced strain (Example 12). HPLC chromatogram of the phytoene fraction obtained from a transgenic strain expressing the CrtM quadruple mutant (E180G, L160W, A245T, G138A) (Example 12). The amount of pinene synthesis when the Dxs wild type or the top seven Dxs mutants (mutA, mutB, mutC, mutD, mutE, mutF, and mutG) selected by FACS analysis were additionally expressed in the pinene-synthesizing strain (pJ404-pT5/lacO-PtpsQ457L-gps) (Example 10). Construct diagram of pJ404-pT5/lacO-PtpsQ457L-gps (Example 10).

(Summary of the Invention)
The present invention relates to a carotenoid having an asymmetric skeleton, a carotenoid having a fluorescent asymmetric skeleton, a method for producing a carotenoid having a fluorescent asymmetric skeleton, a method for screening for a mutant diapophytoene synthase, and a method for screening an enzyme involved in a carotenoid precursor supply pathway.
The carotenoid of the present invention includes both the carotenoid having an asymmetric skeleton of the present invention and the carotenoid having a fluorescent asymmetric skeleton of the present invention.
Furthermore, the method for producing a carotenoid of the present invention includes the method for producing a carotenoid having an asymmetric skeleton of the present invention and/or the carotenoid having a fluorescent asymmetric skeleton of the present invention.

(Carotenoids with asymmetric skeletons)
The "carotenoid having an asymmetric skeleton" of the present invention is a carotenoid having an X chain and a Y chain, and having three conjugated double bond structures (three conjugated double bond structures present at the connection between the X chain and the Y chain), where X is a structure in which n isoprenoid units (C5) are linearly bonded, Y is a structure in which m isoprenoid units (C5) are linearly bonded, the terminal carbon of the X chain forms a double bond with the terminal carbon of the Y chain to form three conjugated double bond structures, and n and m are integers of 1 to 8, but are different from each other.
Specifically, the carotenoids are represented by chemical formulas 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A and 10A shown in FIG. 1 and FIG. 31.

(Carotenoids with fluorescent asymmetric skeletons)
The "carotenoid having a fluorescent asymmetric skeleton" of the present invention is a carotenoid having an X chain and a Y chain and a structure having five conjugated double bonds, where X is a structure in which n isoprenoid units (C5) are linearly bonded, Y is a structure in which m isoprenoid units (C5) are linearly bonded, the terminal carbon of the X chain forms a double bond with the terminal carbon of the Y chain to form a structure having five conjugated double bonds, and n and m are integers from 1 to 8, but are different from each other.
More specifically, a single bond adjacent to three conjugated double bond structures present at the link between the X chain and the Y chain (particularly, a single bond on the X chain side) is desaturated. The "carotenoid having a fluorescent asymmetric skeleton" of the present invention has five conjugated double bond structures due to the desaturation, and further has fluorescence (fluorescence ability).
In the present invention, the fluorescence is not particularly limited in terms of fluorescence intensity, and it is sufficient that the fluorescence can be detected by a method for confirming the fluorescence of a compound known per se (e.g., excitation wavelength: 352 nm, fluorescence wavelength: 484 nm).
Specifically, the carotenoids are represented by chemical formulas 1B, 2B, 3B, 4B, 5B, 6B, 7B, 8B, 9B and 10B shown in FIG. 1 and FIG. 31.

(Carotenoid of the present invention)
Preferred examples of the carotenoid of the present invention include the following.

(Method for producing the "carotenoid having an asymmetric skeleton" of the present invention)
The method for producing the "carotenoid having an asymmetric skeleton" of the present invention is not particularly limited as long as it can produce a carotenoid having an X chain and a Y chain and having three conjugated double bond structures (three conjugated double bond structures present at the connecting portion between the X chain and the Y chain).
Specifically, there are no particular limitations as long as the system can carry out the following synthetic pathway (steps) (e.g., chemical synthesis system, cell synthesis system, etc.).
A process of condensing two molecules of prenyl diphosphate with different sizes (a prenyl diphosphate having n isoprenoid units (X chain) and a prenyl diphosphate having m isoprenoid units (Y chain)).

(Method for producing the "fluorescent carotenoid having an asymmetric skeleton" of the present invention)
The method for producing the "carotenoid having a fluorescent asymmetric skeleton" of the present invention is not particularly limited as long as it can produce a carotenoid having an X chain and a Y chain and five conjugated double bond structures (single bonds adjacent to three conjugated double bond structures present at the link between the X chain and the Y chain are desaturated. More specifically, only the single bond on the X chain side is substantially unsaturated.
Specifically, there are no particular limitations as long as the system can carry out the following synthetic pathway (steps) (e.g., chemical synthesis system, cell synthesis system, etc.).
A: a step of desaturating single bonds adjacent to three conjugated double bond structures present at the junction between X and Y chains of a carotenoid having an X chain and a Y chain and three conjugated double bond structures (three conjugated double bond structures present at the junction between X and Y chains); or B: 1) a step of condensing two molecules of prenyl diphosphates having different sizes (a prenyl diphosphate (X chain) having n isoprenoid units and a prenyl diphosphate (Y chain) having m isoprenoid units), and 2) a step of desaturating single bonds adjacent to three conjugated double bond structures present at the junction between X and Y chains of the carotenoid produced in 1).

(Carotenoid synthesis pathway)
The biosynthetic pathway of carotenoids is explained below. In nature, carotenoids are biosynthesized from mevalonate or pyruvate. First, isopentenyl diphosphate (hereinafter also referred to as "IPP") and dimethylallyl diphosphate (hereinafter also referred to as "DMAPP"), which is an isomerized compound of IPP, are synthesized by the mevalonate pathway or the non-mevalonate pathway. Next, IPP and DMAPP are condensed to synthesize geranyl diphosphate (hereinafter also referred to as "C10PP"), and two more IPPs are added sequentially to synthesize farnesyl diphosphate (hereinafter also referred to as "C15PP") and geranylgeranyl diphosphate (hereinafter also referred to as "C20PP").
C15PP is synthesized by farnesyl diphosphate synthase, and C20PP is synthesized by geranylgeranyl diphosphate synthase.

In the pathway for synthesizing 40-carbon carotenoids, phytoene synthase (CrtB) condenses two molecules of C20PP to synthesize phytoene, which becomes the precursor of carotenoids (the carotenoid skeleton compound).
Furthermore, phytoene is successively desaturated to synthesize phytofluene, zeta-carotene, neurosporene, lycopene, tetradehydrolycopene, etc. The terminal end of lycopene is modified by cyclization or oxidation to synthesize various carotenoids such as alpha-carotene, beta-carotene, gamma-carotene, delta-carotene, epsilon-carotene, lutein, zeaxanthin, canthaxanthin, fucoxanthin, astaxanthin, antheraxanthin, violaxanthin, etc.

(Method for producing carotenoid of the present invention)
The method for producing the carotenoid of the present invention can utilize the above-mentioned known carotenoid biosynthetic pathway, for example. More specifically, the carotenoid of the present invention can be produced by modifying the biosynthetic pathway of a carotenoid present in nature.
Therefore, enzymes and compounds known per se can be used, except for "an enzyme that condenses two molecules of prenyl diphosphate with different sizes (a mutant of carotenoid synthase (CrtM): modified diapophytoene synthase)" and "an enzyme that desaturates a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain of a carotenoid having an asymmetric skeleton (a mutant of phytoene desaturase (CrtI)) and the like" which will be described below.

(An enzyme that condenses two molecules of prenyl diphosphate of different sizes)
The "enzyme capable of bicondensing two different sized prenyl diphosphate molecules" used in the method for producing carotenoids of the present invention is not particularly limited as long as it is a carotenoid synthesis enzyme (e.g., CrtM) modified so as to be capable of bicondensing two different sized prenyl diphosphate molecules. In addition, it may be derived from any organism, including plants, bacteria, etc.

In the present invention, the "enzyme gene capable of bicondensing two differently sized prenyl diphosphate molecules" encodes an "enzyme capable of bicondensing two differently sized prenyl diphosphate molecules." The "enzyme gene capable of bicondensing two differently sized prenyl diphosphate molecules" of the present invention has a mutation introduced therein, as compared with the wild type, in order to achieve the purpose of bicondensing two differently sized prenyl diphosphate molecules.
For example, CrtM (F26A, W38A) used in Example 1 means that the 26th amino acid of wild-type CrtM (SEQ ID NO: 1) has been mutated from F to A, and the 38th amino acid has been mutated from W to A.
The "enzyme gene that condenses two molecules of prenyl diphosphate with different sizes" can be isolated and identified by preparing a library of mutant genes, screening the library for a gene encoding an enzyme with a desired function, and determining the nucleotide sequence. The mutant CrtM gene can be obtained by chemical synthesis, PCR using a cloned probe as a template, site-directed mutagenesis, etc.
In this specification, the term "gene (DNA molecule)" is intended to include not only double-stranded DNA but also each of the single-stranded DNAs constituting the double-stranded DNA, that is, the sense strand and the antisense strand, and is not limited in length. Therefore, unless otherwise specified, the gene (polynucleotide) encoding the amino acid sequence of the present invention includes double-stranded DNA including genomic DNA and single-stranded DNA including cDNA (sense strand), as well as single-stranded DNA (antisense strand) having a sequence complementary to the sense strand, synthetic DNA, and fragments thereof.

The present inventors have confirmed, using a screening method known per se, that the following enzymes are capable of bicondensing two prenyl diphosphate molecules of different sizes (see Figures 30 and 31).
CrtM (F26A, W38A): C20PP + C15PP = C35PP
CrtM (F22A, F26A, W38A, E180G, F233A): C20PP + C30PP = C50PP
CrtM (F26A, W38A, I241A): C20PP + C25PP = C45PP
CrtM (F22A, W38A, E180G): C15PP + C30PP = C45PP
CrtM (L160W) or (L160W, E180G, A245T): C15PP + C10PP = C25PP
CrtM (F26A, W38A): C15PP + C25PP = C40PP
CrtM (L160W, E180G, A245T): C15PP + C5PP = C20PP

In addition, the following enzymes can condense two prenyl diphosphate molecules of different sizes (see Figure 31).
CrtM (F26A, W38A, L145A, E180G, F233A): C15PP + C35PP = C50PP
CrtM (F26A, W38A, L145A, E180G, F233A): C20PP + C35PP = C55PP
CrtM (F26A, W38A, L145A, E180G, F233A): C25PP + C35PP = C60PP

(An enzyme that desaturates the single bonds adjacent to the three conjugated double bonds at the junction between the X and Y chains of carotenoids with asymmetric skeletons.)
The "enzyme that desaturates single bonds adjacent to three conjugated double bond structures present at the link between the X chain and the Y chain of a carotenoid having an asymmetric skeleton" used in the method for producing a carotenoid of the present invention is not particularly limited as long as it can desaturate single bonds adjacent to three conjugated double bond structures present at the link between the X chain and the Y chain of a carotenoid having an asymmetric skeleton of the present invention. In addition, it may be derived from any organism, including plants, bacteria, etc.

In the present invention, the "enzyme gene that desaturates a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain of a carotenoid having an asymmetric skeleton" encodes an "enzyme that desaturates a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain of a carotenoid having an asymmetric skeleton". The "enzyme that desaturates a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain of a carotenoid having an asymmetric skeleton" of the present invention may have a mutation introduced therein, compared to the wild type, in order to achieve the purpose of desaturating a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain of a carotenoid having an asymmetric skeleton (particularly, substantially desaturating only a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain of a carotenoid having an asymmetric skeleton (particularly, a single bond in the X chain)).
For example, CrtI (E69K, R152C, F419L) used in Example 2 means that the 69th amino acid of wild-type CrtI (SEQ ID NO: 2) has been mutated from E to K, the 152nd amino acid has been mutated from R to C, and the 419th amino acid has been mutated from F to L.
The above enzyme can be isolated and identified by preparing a library of mutant genes, screening the library for genes encoding enzymes with the desired functions, and determining the nucleotide sequences. Mutant CrtI genes can be obtained by chemical synthesis, PCR using a cloned probe as a template, site-directed mutagenesis, etc.

The present inventors have confirmed, using a screening method known per se, that the following enzymes can substantially desaturate only the single bonds adjacent to the three conjugated double bond structures present at the junction between the X and Y strands (particularly, the single bonds on the X strand side) (see Figs. 1 and 31).
PDS (SEQ ID NO: 3): C20PP + C15PP = C20 side of C35PP (X chain)
CrtI (E69K, R152C, F419L): C20PP + C30PP = C20 side of C50PP (X chain)
CrtI (E69K, R152C, F419L): C20PP + C25PP = C20 side of C45PP (X chain)
CrtN (L155Q): C15PP + C30PP = C15 side of C45PP (X chain)
CrtI: C15PP + C10PP = C15 side of C25PP (X chain)
CrtN (L155Q): C15PP + C25PP = C15 side of C40PP (X chain)
CrtN (L155Q): C15PP + C5PP = C15 side of C20PP (X chain)

In addition, the following enzymes can desaturate the single bonds adjacent to the three conjugated double bonds at the junction between the X and Y strands (especially the single bonds on the X strand side) (see Figures 1 and 31).
CrtN (L155Q): C15PP + C35PP = C15 side of C50PP (X chain)
CrtI (E69K, R152C, F419L): C20PP + C35PP = C55PP C20 side (X chain)
Step number mutant of CrtIp (SEQ ID NO: 5): C25PP + C35PP = C25 side of C60PP (X chain)
The "property of substantially desaturating only a single bond (particularly, a single bond on the X-chain side)" of the enzyme means that the target compound can be synthesized by substantially desaturating only a single bond (particularly, a single bond on the X-chain side) in the synthesis system described below, and does not mean that no other by-products are produced. In the present invention, the "enzyme that substantially desaturates only a single bond (particularly, a single bond on the X-chain side) adjacent to three conjugated double bond structures present at the link between the X-chain and the Y-chain" is preferably an enzyme that uses a carotenoid having an asymmetric skeleton with three conjugated double bond structures present at the link between the X-chain and the Y-chain as a substrate, catalyzes a reaction that desaturates the single bond on the X-chain side of the single bonds adjacent to the three conjugated double bond structures present at the link between the X-chain and the Y-chain, and produces a carotenoid having a fluorescent asymmetric skeleton with X-chain and Y-chain and five conjugated double bond structures in a separable and extractable ratio (preferably, as a main product).

(Prenyl diphosphate)
In the present invention, "n or m prenyl diphosphate synthesis genes" are genes that encode "n or m prenyl diphosphate synthases".
In addition, prenyl diphosphates synthesized by endogenous biosynthetic systems in cells such as Escherichia coli can also be used. For example, C15PP is synthesized in the Escherichia coli system.
The "n or m prenyl diphosphate synthase genes" can be isolated and identified by preparing a library of mutant genes, screening the library for genes encoding enzymes having the desired function, and determining the nucleotide sequence. Once the nucleotide sequence has been determined, mutant prenyl diphosphate synthase genes can be obtained by chemical synthesis, PCR using a cloned probe as a template, site-directed mutagenesis, or the like.

The following enzymes are known to be capable of synthesizing prenyl diphosphate, or have been confirmed by the present inventors to be capable of synthesizing prenyl diphosphate using a screening method known per se.
FDST121A: Enzyme that synthesizes C20PP
HexPS: an enzyme that synthesizes C30PP
FDSY81M: Enzyme that synthesizes C20PP
HepSTA107L: An enzyme that supplies C25PP
FDS (SEQ ID NO: 4): Enzyme that supplies C15PP
Gps: Enzyme that supplies C10PP

(Cells that can be used in the method for producing carotenoids of the present invention)
The cells that can be used in the method for producing carotenoids of the present invention can be produced by selecting an appropriate expression vector and using a known method for introducing and expressing a foreign gene (e.g., Sambrook, J., Russel, D. W., Molecular Cloning A Laboratory Manual, 3rd Edition, CSHL Press, 2001). The gene to be introduced into the cells by transformation is prepared by a standard method such as PCR, the gene is incorporated into an expression vector suitable for the host by a standard method, the desired vector is selected, and the host cell is transformed with the vector by a standard method. When transforming cells with two or more types of genes, the multiple genes may be incorporated into the same expression vector for transformation, or may be incorporated into different expression vectors for co-transformation.

There are no limitations on the host cells, but in consideration of shortening the culture time and ease of cloning, microorganisms such as Escherichia coli, Bacillus subtilis, and yeast are preferred. In particular, Escherichia coli and yeast are preferred. Suitable Escherichia coli include cloning strains such as Escherichia coli XL1-Blue (hereinafter simply referred to as "E. coli XL1-Blue"), expression strains such as HB101 and BL21, as well as gene knockout strains that synthesize large amounts of terpene precursors, such as JW1750 ΔgdhA (glutamate dehydrogenase deficient) and J W0110 ΔaceE (pyruvate dehydrogenase deficient) (Baba, T. et al.; Mol Syst Biol 2, 2006 0008 (2006)), and suitable yeasts include standard budding yeast, INVSc1 (invitrogen), YPH499 (stratagene), etc.

Host cells that can be used include E. coli, insect cells, yeast cells, plant cells, and the like.
The host cell has the following genes (1) to (4) introduced therein or is endogenous thereto:
(1) a gene for synthesizing a prenyl diphosphate having n isoprenoid units; (2) a gene for synthesizing a prenyl diphosphate having m isoprenoid units; (3) an enzyme gene for condensing two molecules of a prenyl diphosphate having n isoprenoid units and a prenyl diphosphate having m isoprenoid units; and (4) an enzyme gene for desaturating a single bond adjacent to three conjugated double bonds present at the junction between the X and Y chains.

In addition, preferred combinations of synthetic enzyme genes to be introduced or present in each cell, as necessary, are as follows, but are not particularly limited.
E. coli: IDI, crtE, crtB, crtI (or mutant), crtY, crtZ, ZEP, AtCYO1, NsRF, gdh
Yeast system: IDI, crtE, crtB, crtI (or mutant), crtY, crtZ, ZEP, AtCYO1, NsRF, gdh
Insect system: IDI, crtE, crtB, crtI (or mutant), crtY, crtZ, ZEP, AtCYO1, NsRF, gdh
Plant system: IDI, ZEP, AtCYO1, NsRF, gdh IDI: isopentenyldiphosphate (IPP) isomerase crtE: GGPP synthase crtB: phytoene synthase crtI: phytoene desaturase crtY: lycopene β-cyclase crtZ: β-carotene hydroxylase ZEP: zeaxanthin epoxidase AtCYO1: chaperone protein NsRF: electron transport protein gdh: NADPH regeneration protein

The expression vector into which the gene is to be inserted is not particularly limited and may be a commonly used vector, for example, pUC18, pACYC184, etc. derived from the host when Escherichia coli is used, pUB110, pE194, pC194, pHY300PLK DNA, etc. derived from the host when Bacillus subtilis is used, and pRS303, YEp213, TOp2609, etc. derived from the host when yeast is used.
Whether or not the gene of interest has been introduced into the host cell can be confirmed by a standard method, for example, PCR, Southern hybridization, Northern hybridization, or the like.

The method for producing carotenoids of the present invention includes a step of culturing the transformant cells obtained as described above in a medium. The medium may be any medium that contains a substance that can be a source of carotenoid skeletal compounds, and may be a medium that contains components that are generally used in cell culture. In cells in which carotenoid skeletal compounds are synthesized through the metabolism of IPP and DMAPP, the medium may contain a carbon source that can be a source of IPP and DMAPP. Examples of such carbon sources include various sugars such as glucose.

The temperature during cultivation is not particularly limited, but is preferably 18 to 30°C, and more preferably 20 to 30°C. The cultivation time is also not particularly limited, but cultivation is preferably carried out for 12 to 72 hours from the expression of the gene introduced by transformation, and more preferably for 24 to 48 hours. The carotenoid of the present invention can be recovered from the culture after cultivation according to a method commonly used for obtaining products such as carotenoids from cells of microorganisms, etc. The carotenoid may be obtained from the cells by isolating only the cells from the culture.

The results of this Example below show that if a polar organic solvent, such as dodecane, decane, or isopropyl myristate (particularly dodecane), is added to the medium of cultured cells that synthesize small carotenoids such as C25 carotenoids (C15+10-phytofluene) and C20 carotenoids (C15+5-phytofluene), the small carotenoids are extracted into the organic solvent phase, making it possible to produce the carotenoids of the present invention while continuously extracting them without accumulating within the cultured cells.

(Uses of the carotenoid of the present invention)
As will be apparent from the following Examples, the carotenoids of the present invention (particularly, carotenoids having a fluorescent asymmetric skeleton) and their synthetic pathways can be used for screening of mutant diapophytoene synthases, mutant phytoene desaturases, and enzymes involved in the carotenoid precursor supply pathway (e.g., DXS, DXR, IspG, IspH), etc. Here, DXS, DXR, IspG, and IspH respectively mean the following enzymes.
DXS: 1-deoxy-D-xylulose-5-phosphate synthase
DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase
IspG: 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase
IspH: (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate reductase
These enzymes are key enzymes in the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway and contribute to isoprenoid synthesis.

(Screening of mutant diapophytoene synthase)
The "screening method for mutant diapophytoene synthase" of the present invention comprises the following steps (1) and (2).
(1) introducing a mutation-introduced diapophytoene synthase gene library (a gene library in which substitutions, deletions, insertions and/or additions have been introduced into a wild-type diapophytoene synthase gene) into a cell capable of synthesizing a carotenoid having an X chain and a Y chain and having five conjugated double bond structures from a carotenoid having an X chain and a Y chain and having three conjugated double bond structures;
(2) selecting cells expressing the mutant diapophytoene synthase using the fluorescence intensity of the cells as an indicator.
Furthermore, if a carotenoid having a fluorescent asymmetric skeleton of the present invention is synthesized, the fluorescence intensity increases.
In particular, the screening method can determine the function of a mutant diapophytoene synthase in terms of its ability to synthesize a carotenoid having a fluorescent asymmetric skeleton, and further whether said ability is higher, compared to a wild-type diapophytoene synthase.

The "screening method for enzymes involved in the carotenoid precursor supply pathway" of the present invention comprises the following steps (1) and (2).
(1) introducing a gene library (a gene library in which substitutions, deletions, insertions and/or additions have been introduced into wild-type genes encoding enzymes involved in the carotenoid precursor supply pathway) into cells capable of synthesizing a carotenoid having an X chain and a Y chain and having five conjugated double bond structures from a carotenoid having an X chain and a Y chain and having three conjugated double bond structures, into cells capable of synthesizing a carotenoid having an X chain and a Y chain and having five conjugated double bond structures from a carotenoid having an X chain and a Y chain and having three conjugated double bond structures;
(2) Culturing the cells into which the gene has been introduced, and then selecting cells expressing an enzyme involved in the mutant carotenoid precursor supply pathway using the fluorescence intensity of the cells as an indicator.
Furthermore, if a carotenoid having a fluorescent asymmetric skeleton according to the present invention is synthesized, the fluorescence intensity increases.
In particular, the screening method can determine whether the function of an enzyme involved in a mutant carotenoid precursor supply pathway is the ability to synthesize carotenoids having a fluorescent asymmetric skeleton, and even whether said ability is enhanced, compared to an enzyme involved in a wild-type carotenoid precursor supply pathway.

The fluorescence intensity can be measured by a method known per se.
In addition, the cells used may be the "cells that can be used in the method for producing a carotenoid of the present invention" described above.
Furthermore, the "gene mutation" in the mutated enzyme gene library can be performed using a method for introducing mutations that is publicly known per se (e.g., random mutation, site-specific amino acid substitution, partial deletion of amino acid residues, or insertion of a peptide or other protein).

(Mutant diapophytoene synthase)
Based on the results obtained in Example 7 below, the "mutant diapophytoene synthase" of the present invention is an enzyme involved in the synthesis of a carotenoid having an asymmetric skeleton of the present invention (an enzyme that condenses an X chain and a Y chain, each of which has an isoprenoid unit), and has any one of the following mutations (1) to (4) (the amino acid mutations are shown in parentheses) compared with wild-type CrtM (SEQ ID NO: 1):
(1) A151G (I51G) and G733A (A245T);
(2) A233T(H78L), A464G(D155G) and T500C(I167T);
(3) C274T(H92T), A339G, and T500C(I167T); or (4) one, two, or three mutations selected from (L160W), (E180G), and (A245T).

In addition, the "mutated diapophytoene synthase" of the present invention also includes derivatives of the enzyme (protected derivatives, glycosylated derivatives, acylated derivatives, or acetylated derivatives).
The protected derivatives, glycosylated derivatives, acylated derivatives, or acetylated derivatives can be obtained by methods known per se.

The present invention will be explained in detail below with reference to examples, but the present invention is not limited to these examples.

(Synthesis of C20+15-phytofluene)
Phytoene having a C35 skeleton can be biosynthesized by head-to-head condensation of C20PP (geranylgeranyl diphosphate) and C15PP (farnesyl diphosphate) (Reference: D. Umeno, and F. H. Arnold, A C35 Carotenoid Biosynthetic Pathway., Appl Environ Microb., 69 3573-3579 (2003)). In this example, a C20+15 carotenoid was synthesized with reference to the reference.
The inventors tried various combinations of size mutants of CrtM derived from Staphylococcus aureus and farnesyldiphosphate synthase (FDS) derived from the moderately thermophilic bacterium Bacillus stearothermophilus, and constructed an E. coli system that selectively produces this C35 skeleton phytoene (reference: M. Furubayashi et al., A highly selective biosynthetic pathway to non-natural C50 carotenoids assembled from moderately selective enzyme, Nat. Commun., 6 7534 (2015)).
In the co-expression system of the two CrtM/FDS mutant pairs obtained here, phytoene desaturase PDS (derived from Synechococcus elongatus) was additionally expressed. Colonies that showed faint fluorescence in response to black light were obtained (Fig. 2(b)). When the culture medium was centrifuged, the resulting pellet showed fluorescence.
C15PP is synthesized in Escherichia coli. FDST121A is an enzyme that synthesizes C20PP, CrtMF26A and W38A are enzymes involved in the head-to-head condensation of C20PP and C15PP, and PDS is a desaturase of phytoene.
When these cells were disrupted and extracted with acetone, a large absorption peak derived from phytofluene was observed in the lipid-soluble components (Fig. 3(a)). This indicates that the majority of the carotenoid pigment is phytofluene. The fluorescence spectrum of these components was very similar to that of phytofluene previously reported (λex = 484 nm, λem = 484 nm, see, for example, P. O. Andersson, et. al., Photophysical Characterization of Natural cis-Carotenoids, Photochem. Photobiol., 74, 549-557 (2001)) (Fig. 3(b)).
The extract was analyzed by HPLC, as shown in Figure 4(b). From the molecular weight (a 2H molecular weight reduction due to one desaturation site), elution time (polarity), and absorption spectrum (334 nm, 349 nm, 367 nm: Figure 4(d)), it was found that PDS gave C35-phytofluene. The high size specificity of PDS was observed. On the other hand, PDS did not give any desaturation products to C30 phytoene skeleton (Figure 4(a)), but for C40, because it has two C20 ends, it gave γ-carotenoid through two steps (Figure 4(c)).
As described above, a carotenoid having five conjugated double bond structures represented by formula (2B) could be synthesized by the method for producing a carotenoid of the present invention.

(Synthesis of C30+20-phytofluene)
In this example, the carotenoid synthesis system of Example 1 was used, but the plasmid expression/construct shown in FIG. 5(a) was used.
HexPS is an enzyme that synthesizes (supplies) C30PP, fdsY81M is an enzyme that synthesizes (supplies) C20PP, CrtMF22A, F26A, W38A, E180G, and F233A are enzymes involved in the head-to-head condensation of C30PP and C20PP, and CrtIE69K, R152C, and F419L are single desaturases on the C20 side (X chain). HexPS is a heterodimer and is composed of the subunits HexA (SEQ ID NO: 7) and HexB (SEQ ID NO: 8).
From the fluorescence confirmation of the obtained pellet, the MS peak (a 2H molecular weight reduction due to one desaturation site) and the absorbance, it was confirmed that peak 2 in Figure 5(b) was C30+20-phytofluene.
As described above, in this example, a carotenoid having a five-conjugated double bond structure represented by formula (5B) was synthesized as the main component.

(Synthesis of C25+20-phytofluene)
In this example, the carotenoid synthesis system of Example 1 was used, but the plasmid expression/construct shown in FIG. 6(a) was used.
The HepSTA107L mutant is a size-specific mutant of HepST that supplies C25PP, fdsY81M is an enzyme that synthesizes (supplies) C20PP, CrtMF26A, W38A, and I241A are enzymes involved in the head-to-head condensation of C25PP and C20PP, and CrtIE69K, R152C, and F419L are single desaturases on the C20 side (X chain) (T. Koyama et al., Inter-subunit Location of the Active Site of Farnesyl Diphosphate Synthase: Reconstruction of Active Enzymes by Hybrid-Type Heteromeric Dimers of Site-Directed Mutants, Biochemistry, 39, 463-469 (2000)). HepST is a heterodimer and is composed of HepS (SEQ ID NO: 9) and HepT (SEQ ID NO: 10).
From the fluorescence confirmation of the obtained pellet, the MS peak (a molecular weight reduction of 2H due to one desaturation site) and the absorbance, it was confirmed that peak 2 in FIG. 6(b) was C25+20-phytofluene.
As described above, in this example, a carotenoid having a five-conjugated double bond structure represented by formula (8B) was synthesized.

(Synthesis of C30+15-phytofluene)
In this example, the carotenoid synthesis system of Example 1 was used, but the plasmid expression/construct shown in FIG. 7(a) was used.
In addition, HexPS is an enzyme that supplies C30PP, fds is an enzyme that supplies C15PP, CrtMF22A, W38A, and E180G are enzymes involved in the head-to-head condensation of C30PP and C15PP, and CrtNL155Q is a single desaturase on the C15 side (X chain). The wild-type sequence of CrtN is shown in SEQ ID NO:6.
A single desaturation (only on the C15 side) of the C30+C15=C45 carotenoid backbone was achieved.
From the fluorescence confirmation of the obtained pellet, the MS peak (a 2H molecular weight reduction due to one desaturation site) and the absorbance, it was confirmed that peak 2 in FIG. 7(b) was C30+15-phytofluene.
As described above, in this example, a carotenoid having a five-conjugated double bond structure represented by formula (9B) was synthesized.

(Synthesis of C15+10-phytofluene)
In this example, the carotenoid synthesis system of Example 1 was used, but the plasmid expression/construct shown in FIG. 8(a) was used.
A CrtML160W mutant was created in which the L160 position forming the floor of the S2 site of CrtM was replaced with the bulky amino acid tryptophan, and CrtI was coexpressed with GPS (SEQ ID NO: 11). Here, in order to increase sufficient carotenoid production, Dxs mutants and IspH mutants (WO2022/102763A1) were additionally expressed. Since carotenoid production can be increased by increasing the supply of terpene precursors, the production of C15+10 phytoene can also be improved by additionally expressing other enzymes in the Mep pathway instead of or in addition to Dxs or IspH, introducing enzymes in the mevalonate pathway, or adding a raw material supply pathway from the pentasaccharide pathway by additionally expressing a RibB mutant (Zhou et al., ACS Synth. Biol., 2024, vol.13, pp.876-887).
GPS is an enzyme that supplies C10PP, the CrtML160W mutant is an enzyme involved in the head-to-head condensation of C15PP and C10PP, and CrtI is a single desaturase on the C15 side (X chain).
The construct shown in FIG. 8 achieved a single desaturation of the C10+C15=C25 carotenoid backbone (only the C15 side, which is the X chain).
From the fluorescence of the obtained pellet, the MS peak (a 2H molecular weight reduction due to one desaturation site) and the absorbance, peak 2 was identified as C15+10-phytofluene.
As described above, in this example, a carotenoid having a five-conjugated double bond structure represented by formula (1B) was synthesized.

(Extraction of carotenoids with organic solvents)
Naturally occurring carotenoids are known to accumulate in cell membranes due to their large molecular size and C40 or C30 backbone.
On the other hand, the C25 carotenoid (C15+10-phytofluene) synthesized in Example 5 above is smaller than naturally occurring carotenoids and can therefore permeate cell membranes.
As is clear from the results in Figure 9(c), when the C25 carotenoid biosynthetic strain constructed in Example 5 (Figure 9(a)) was cultured in a medium overlaid with an organic solvent (dodecane), the organic solvent layer turned yellow. Analysis of the lipid-soluble components contained in the organic solvent layer and the cell pellet (Figure 9(b)) revealed that C30 and C25 carotenoids were detected in the cell pellet. On the other hand, essentially only C25 carotenoid (C15+10-phytofluene) was detected in the organic solvent layer.

The results of this example confirmed that if an organic solvent such as dodecane is added to a system (culture medium) that synthesizes small carotenoids such as C25 carotenoids (C15+10-phytofluene), the small carotenoids are extracted into the organic solvent phase, making it possible to produce small carotenoids while continuously extracting them without them accumulating within the cells.

(Method of screening for enzymes that condense X and Y to synthesize carotenoids having an asymmetric skeleton (X≠Y))
It has been confirmed from the above Examples 1 to 5 that fluorescent (phytofluene or pentaene) carotenoids can be produced by co-expressing a carotenoid desaturase with an asymmetric carotenoid. In other words, by co-expressing an appropriate carotenoid desaturase, the formation of a fluorescent asymmetric carotenoid can be selectively detected by fluorescence.
For example, in Example 5, fluorescent carotenoids were formed by additionally expressing CrtI on the C25 (C10+C15) backbone (Figure 8). On the other hand, only highly unsaturated non-fluorescent carotenoid pigments accumulated in C30 (15+15). Based on this novel finding, mutants that preferentially (selectively) synthesize C25 (C10+C15) rather than C30 (15+15) under CrtI co-expression conditions can be selected using fluorescence as an indicator.
In this example, we therefore screened for an enzyme (mutant diapophytoene synthase) that condenses X and Y chains to synthesize carotenoids with an asymmetric skeleton (X≠Y). A previous study reported that CrtM has a mutation that improves structural stability; the global suppressor mutation E180G (D. Umeno et al., Evolution of the C30 Carotenoid Synthase CrtM for Function in a C40 Pathway, J. Bacteriol., 184, 6690-6699 (2002)).
In this Example, therefore, we created a CrtML160W,E180G mutant by introducing E180G into the CrtML160W mutant, and prepared a gene library by randomly introducing mutations into this double mutant crtML160W,E180G gene using the ep-PCR method (P. C. Cirino, et al., Generating mutant libraries using error-prone PCR, Methods Mol. Biol., 231, 3-9 (2003)).
The constructed gene library was introduced into E. coli together with the C10PP synthase gene (ispA(S80F); a mutant of ispA derived from Escherichia coli; Chen et al., J. Ind. Microbiol. Biotechnol. vol.43, pp.1281-1292), CrtI, DxsmutC, and IspHmut12, and colonies were formed (FIG. 10(a)). When the formed colonies were irradiated with ultraviolet light, 14 mutants were confirmed to emit more fluorescence than the parent CrtML160W, E180G mutants (FIG. 10(b)). The Dxs and IspH mutants were used to increase carotenoid production, and similar effects can be obtained by introducing enzymes of the Mep pathway or the mevalonate pathway, as in Example 5.

These mutants were isolated, and the plasmids were recovered and then reintroduced into fresh E. coli (2nd screening). They were expressed together with CrtI, C10PP supply enzyme, DxsmutC and IspHmut12, and cultured in a medium overlaid with dodecane as shown in Example 6. After the culture was completed, the dodecane phase and cell pellet were collected, and carotenoids were extracted from the cell pellet using acetone (Figure 11(c)). The fluorescence intensity of the collected dodecane phase (Figure 11(a)) and the amount of carotenoid synthesis contained in acetone (Figure 11(b)) were measured.
Comparing the fluorescence intensity of the dodecane phase of the parent CrtML160W and E180G, all mutants except mut7, mut9, mut13, and mut14 showed increased fluorescence intensity compared to the parent (Fig. 11(a)). The reason why fluorescence was not observed in mut14 is thought to be due to contamination of the mutant due to adjacent colonies when the mutant was recovered. For reference, the absorption spectrum (400 nm) of the dodecane phase was also measured, and the amount of C15+10-ζ-carotene contained in the dodecane phase was also measured from the absorption spectrum. When CrtI is given the C15+10 substrate, it also synthesizes C15+10-ζ-carotene (2-step desaturated product) at the same level as C15+10-phytofluene (1-step desaturated product) (Fig. 8). In fact, the amount of C15+10-ζ-carotene detected in the dodecane phase generally correlated with the phytofluene fluorescence intensity in the dodecane phase (Figure 11(b)).
Comparing the amount of carotenoid synthesis in the cell pellet, mut3, mut5, mut6, mut7, mut9, mut12 and mut13 synthesized more than twice as much carotenoid as the parent (Figure 11(c)). Mutants mut8 and mut10, which have particularly high phytofluene fluorescence intensity in the dodecane phase but do not synthesize as much carotenoid in the cell pellet as mut-3/5/6/7/9/12/13, are expected to be mutants that selectively synthesize C15+10-phytoene. Similarly, mut2, which synthesizes about half the amount of carotenoid in the cell pellet as the parent, was selected because it is thought to be a mutant with a more shifted size specificity.

To evaluate the C15+10-phytoene synthesis activity of the selected CrtM mutant, the CrtM mutant, C10PP supplying enzyme, DxsmutC and IspHmut12 were co-expressed and cultured, and product analysis was performed (Figure 12).
The amount of C15+10-phytoene synthesis in mut8 and mut10 was 1.3-fold higher than in the parent. Furthermore, the amount of C30-phytoene synthesis was comparable to that in the parent. Only mut2 showed a 1.1-fold increase in C15+10-phytoene synthesis compared to the parent. Furthermore, the amount of C30-phytoene synthesis in mut2 was reduced to about 10% of that in the parent. As expected from the second screening, it was confirmed that mut2 is a mutant that can efficiently synthesize low molecular weight carotenoids (C15+10, C15+5) with a higher size specificity.
Furthermore, the mutation sites of Mut2, Mut8 and Mut10 are shown in FIG.
Structural mapping revealed that the A245T mutation in Mut2 was located at a position likely to affect specificity, so this mutation was introduced alone into the parent CrtM (L160W, E180G) to create CrtM (L160W, E180G, A245T). As a result, it was found that, as in Mut2, the amount of C15+C15 carotenoids synthesized was low, and C15+C10 carotenoids were mainly synthesized. In other words, it was revealed that it was this mutation that caused the specificity shift of Mut2.
As described above, in this Example, a screening method for an enzyme (CrtM mutant) that condenses X and Y chains to synthesize carotenoids with an asymmetric skeleton (X≠Y) was completed. Similarly, various factors that increase the production of C25-phytofluene, such as enzymes and host strains that increase its precursors DMAPP and IPP, can be selected and obtained from a large mutant pool.

(Synthesis of C25+15-phytofluene)
In this example, the carotenoid synthesis system of Example 1 was used, but the Plas mid expression/construct shown in Figure 25(a) was used.
FDSwt is an enzyme that supplies C15PP, HepST mutant (Small Subunit A107L) is an enzyme that supplies C25PP, CrtM (F26A, W38A) is an enzyme involved in the head-to-head condensation of C25PP and C15PP, and CrtNL155Q is a single desaturase on the C15 side (X chain). The HepST mutant is composed of HepS (SEQ ID NO: 9) and HepT (A107L) (SEQ ID NO: 10).
A single desaturation (only on the C15 side) of the C25+C15=C40 carotenoid skeleton was achieved.
Based on the fluorescence of the obtained pellet, the MS peak (a 2H molecular weight reduction due to one desaturation site) and the absorbance, peak-2 in Figure 25(b)(C) was confirmed to be C25+15-phytofluene.
As described above, in this example, a carotenoid having a five-conjugated double bond structure represented by formula (3B) was synthesized.

(Synthesis of C15+5-phytofluene)
CrtM (L160W,E180G,A245T) was co-expressed with the mutant IPK3m (SEQ ID NO:13) of isopentenyl monophosphate kinase IPK, and cultured in the presence of dimethylallyl alcohol (10 mM). This mutant enzyme can diphosphorylate DMAOH and convert it to DMAPP, a C5 isoprenyl unit (AO Chatzivasileiou, V. Ward, S. McBride Edgar, G. Stephanopoulos: PNAS, 116, 506-511 (2018)).
As a result, it was confirmed that C15+5 carotenoid was synthesized in the cells and the amount of accumulated C15+5 carotenoid was significantly increased (Figure 26). In other words, it was confirmed that C15+5 carotenoid represented by formula (10A) was synthesized.
In addition, by treating C15+5 carotenoids with desaturases (such as CrtNL155Q), fluorescent C15+5 carotenoids can be synthesized.

(Method for screening enzymes involved in the carotenoid precursor supply pathway)
Since phytofluene is a fluorescent molecule, the increase or decrease in the amount of phytofluene accumulated in cells can be indirectly measured by the change in the fluorescence intensity of phytofluene. This phytofluene fluorescence can be used to screen for enzymes related to a better carotenoid precursor supply pathway. Therefore, screening was performed for DXS (derived from Escherichia coli, EC: 2.2.1.7), one of the genes in the carotenoid precursor supply pathway. A library of DXS was created using epPCR using evolutionary engineering (primers (Fwd; TTTTGGTCTCtCCAGGCATCAAATAAAACGAAAGG (SEQ ID NO: 14), Rev; AAAGGCGAGATCACCAAGGTAG (SEQ ID NO: 15)) and PCR was performed with Taq polymerase (NEB) in the presence of Mn 2+ ).
Mn2+ was prepared at 10μM and 50μM. Taq polymerase was used at 1.25μL/50μL PCR, and dNTP was prepared at 200μM. The thermal cycle was 95℃ 30 sec → (95℃ 30 sec, 52℃ 30 sec, 68℃ 2 min) 25 cycles → 68℃ 5 min. The PCR product was purified and subjected to restriction enzyme treatment with BamHI and Hind3, and ligated (16℃, 16h) to the vector sequence treated with BamHI and Hind3 by PCR using primers (Fwd; AAGCTTACTAGTaataCTGCAGAGAG (SEQ ID NO: 16), Rev; TTTTGGATCCGTGGTGATGGTGATG (SEQ ID NO: 17)). The ligation product was introduced into E. coli strain BW25113 by electroporation to recover the library plasmid. The library sizes were (Mn2+ 10 μM; 1.2×10 ⁴ , Mn2+ 50 μM; 1.0×10 ⁴ ).
The dxs library thus obtained was co-transfected into E. coli together with the C20+15-phytofluene synthesis gene group (fdsT121A, CrtMF26A, W38A, pds) obtained in Example 1. ¹⁰⁷ of the obtained transformants were analyzed by FACS. As a result, it was confirmed that the fluorescence intensity of the whole transformant group increased when the dxs mutant was expressed (Figure 29).
The median of the group shifted to the right compared to when Dxswt was expressed, indicating that the library contained Dxs mutants with improved activity and that the difference in their activity could be distinguished by FACS analysis. The top 30 mutants analyzed were sorted, and the functions of 7 of them were evaluated. The functional evaluation was performed using pinene, a volatile monoterpene synthesized from carotenoid precursors, as a probe. That is, the plasmids recovered from each were co-introduced into E. coli together with a plasmid expressing pinene synthase (AgPtPS(Q457L); Tashiro et al., ACS Synth. Biol. 2016, vol.5, pp.1011-1020), and cultured at 30°C in TB medium. After 24 hours, the dodecane fraction that had risen to the top of the medium was analyzed by GC-FID to confirm the production amount per medium.
It was confirmed that the amount of pinene synthesis in E. coli coexpressing each of the seven DXS mutants tested with pinene synthase was significantly increased compared to the amount of pinene synthesis in E. coli coexpressing wild-type DXS (Figure 35).

(Screening for new CrtM mutants with superior DMAPP consumption activity)
In Example 9, C15+C5 (C20 type) carotenoids were synthesized in a system in which CrtM (L160W, E180G, A245T) was co-expressed with the mutant IPK3m (SEQ ID NO: 13) of isopentenyl monophosphate kinase IPK. If a CrtM mutant with DMAPP consumption activity superior to that of the triple mutant CrtM (L160W, E180G, A245T) could be obtained, low molecular weight carotenoids such as C15+C5 could be synthesized more efficiently, and this would be highly useful. Therefore, a screening system was constructed that focuses on the fact that DMAPP, a substrate of CrtM, has an inhibitory effect on ATP synthesis and its excessive accumulation suppresses cell proliferation. Specifically, we explored CrtM mutants by utilizing the mechanism by which CrtM mutants with excellent DMAPP-consuming activity efficiently consume DMAPP in the presence of DMAPP (a system in which DMAPP is synthesized from DMAOH by the activity of IPK3m and DMAPP accumulates in transformed cells), thereby restoring the growth of transfected cells (avoiding the inhibition of cell growth due to excessive DMAPP accumulation) and becoming selectively concentrated.
Using a plasmid expressing CrtM (E180G, L160W, A245T) as a parent, the region encoding glycine at position 138 was diversified by site-saturation mutagenesis using degenerate primers that enabled expression of 32 codons, and a library was created.
The primers used here were Fwd sequence: 5'-GGTGTCGCAGGCACGTTNNKGAGGTTCTGACCCCGATCCTGTC-3' (SEQ ID NO: 18), Rev sequence: 5'-CGTGCCTGCGACACCGTAACAATAG-3' (SEQ ID NO: 19) (In the Fwd primer, N represents an A/C/G/T mixture, and K represents a G/T mixture.) The PCR product obtained was circularized by the intramolecular Gibson Assembly method.
The library plasmids constructed above were introduced into E. coli strain XL1-Blue, which had already been transformed with a plasmid expressing IPK mutants (V73I, Y41V, K204G). The cell population of the transformants was cultured for 40 h on LB agar medium supplemented with 8 mM DMAOH. In this culture system, DMAOH is phosphorylated in two steps by the action of the IPK mutants, converting it to DMAPP.
As described above, CrtM mutants with excellent DMAPP-consuming activity consume DMAPP and restore cell growth, so it is expected that transformed cells expressing the desired CrtM mutants will be selectively enriched during culture.
The cells that had been selected for their DMAPP-consuming ability through culture in liquid medium containing DMAOH were seeded onto LB agar medium containing various concentrations of DMAOH (0, 3, 5, 7, 8, 9, 10 mM) and allowed to form colonies (the amount of bacteria was adjusted so that 1,000 colonies would grow on a plate with a diameter of 10 cm). As the number and size of colonies on LB agar medium supplemented with 8 mM DMAOH were different from the parent (CrtM (E180G, 160W, A245T)), colonies that had formed on agar medium supplemented with 8 mM DMAOH were picked.
Eight colonies that grew on the 8 mM medium were picked up, and the crtM gene was cloned and sequenced. As a result, the breakdown of the amino acid residue at position 138 encoded by the eight clones was as follows:
Ala: 2
Ser: 3
Cys: 1
Thr: 1
Gly: 1 (same as wild type)
It was.
The four mutants obtained were isolated and the specific growth rates (relative growth rate with the growth rate at 0 mM DMAOH set at 1) in the presence or absence of DMAOH (8 mM) were compared. As shown below, G138A had the highest specific growth rate.
(Here, the codon encoding Ala at G138A was GCT.)
We compared the mutant with the G138A mutation with the wild-type CrtM and the parent triple mutant (L160W, E180G, A245T), and found that it had improved resistance to cell growth inhibition caused by DMAPP accumulation (Fig. 32). This quadruple mutant had higher DMAPP consumption activity, suggesting that it is a novel CrtW mutant that is useful for improving the productivity of C20 carotenoids and other compounds.
Example 12

(Confirmation of small molecule carotenoid synthesis)
The expression plasmids of each CrtM mutant used in Example 11 were introduced into E. coli strain XL1-Blue. This was cultured in TB medium (10 mL; no DMAOH added) at 30°C for 48 hours. After collecting the cells by centrifugation and removing the medium, the hydrocarbon fraction was extracted with acetone. This was partitioned into two phases with hexane/water, and the hexane component was collected, evaporated to dryness, and then analyzed by HPLC.
The separation column was Shimpack, ODS (C18), and the mobile phase was A layer: MeOH:water = 9:1, B layer: MeOH:IPA = 6:4, 0.2 mL/min, 60 min. A photodiode array (PDA) detector and a mass detector (ionization: APCI) were used for detection.
The target m/z was measured using the SIM(+) mode of MS. At the same time, a chromatogram of the UV absorption (287 nm) characteristic of phytoene was obtained using a PDA absorption detector, and the production amount of each carotenoid molecular species was quantified from each peak area (Figure 33 (top), Figure 34 (left)).
As shown by the second bar from the top in Figure 33, when CrtM (E180G, L160W, A245T) was expressed, a small amount of phytoene with a C20 (C5 + C15) skeleton was synthesized, but when the quadruple mutant CrtM (E180G, L160W, A245T, G138A) obtained in Example 11 was expressed (third bar from the top in the same figure; left in Figure 34), the amount of carotenoid produced decreased and C20 carotenoid was not detected.
Next, the expression plasmids of each CrtM mutant were introduced into E. coli together with the IPK expression plasmid IPK3m (V73I, Y41V, K204G), and cultured in TB medium containing 10 mM DMAOH in the same manner as described above, and HPLC analysis and mass spectrometry were performed using the methods described above (Figure 33 (bottom), Figure 34 (right)).
As a result, as shown by the bars in the fourth and fifth rows from the top in Figure 33, when two CrtM mutants (CrtM(E180G, L160W, A245T) and CrtM(E180G, L160W, A245T, G138A)) were introduced, the production of C20 carotenoids was significantly increased. The increase in the accumulation of C20 carotenoids (C5+C15) was particularly remarkable when CrtM(E180G, L160W, A245T, G138A) was introduced (fifth row from the top in Figure 33; right in Figure 34).
Through this example, we were able to improve the productivity of the world's smallest C20 (C5 + C15)-phytoene, which has a skeleton size half that of natural phytoene.

(General remarks)
The results of this example demonstrate that (1) a biosynthetic pathway for the mass production of fluorescent carotenoids as main products by the action of desaturases on asymmetric skeletons has been established for the first time in the world.
Furthermore, by establishing the biosynthesis of fluorescent carotenoids, (2) the biosynthetic pathway of asymmetric carotenoids with smaller (continuously extractable) C25 skeletons can also be screened using fluorescence output as an indicator, improving its potency and selectivity.
Carotenoids (C30-C50 skeleton) and triterpenoids (C30 skeleton) have large molecular weights, so they accumulate in the cell membrane and cannot be extracted from the cell unless it is disrupted. On the other hand, volatile terpenes, which are also hydrocarbons, can disperse in small amounts in water. For this reason, if dodecane or the like is layered on top of the culture medium, it is continuously extracted, allowing for continuous production without product toxicity or product feedback. This makes it possible to produce them in microorganisms in quantities that are orders of magnitude greater.
The conventional strategy was to mass-produce farnesene with 15 carbon atoms and synthesize C30 squalene by condensing it with a chemical catalyst (WO2011146837; Squalane and isosqualane compositions and methods for preparing the same). This invention shows that C25 "squalene-like" hydrocarbons, which are only C5 units removed, can be biosynthesized while being continuously extracted into the dodecane phase. This makes it possible to directly mass-produce linear hydrocarbons with value as antioxidants, adjuvants (immunostimulants), cosmetic base materials, special lubricants, and biofuels in microorganisms.

This example confirmed the usefulness of fluorescent carotenoids (phytofluene-type carotenoids) in metabolic engineering.
Conventionally, screening of mutants that increase the titer of the isoprenoid upstream pathway has been performed using the accumulation of carotenoid pigments such as lycopene as an index (C. Wang, et al., Directed Evolution of Metabolically Engineered Escherichia coli for Carotenoid Production, Biotechnol. Prog. 16, 922-926 (2000)). In this method, the degree of coloration due to pigment accumulation in colonies formed on a medium is compared, so the number of colonies that can be formed on an agar medium (up to 10 ⁴ ) is the limit of the screening size. On the other hand, in FACS sorting using phytofluene fluorescence, it is easy to screen 10 ⁷ cells. Furthermore, carotenoid pigments such as lycopene accumulate in the cell membrane, and there is an upper limit to their accumulation capacity. And their membrane accumulation is extremely toxic. For this reason, there is a limit to the amount of pigment accumulation per cell, and when the upstream pathway is improved to a certain extent, the colony color due to pigment accumulation no longer reflects the superiority or inferiority of the precursor pathway. In the case of FACS sorting, single cells can be sorted at an early stage before accumulation saturation is reached, making screening with a wide dynamic range possible.

The present invention can provide a carotenoid having an asymmetric skeleton. In addition, the present invention can provide a carotenoid having a fluorescent asymmetric skeleton, a method for producing a carotenoid having a fluorescent asymmetric skeleton, a method for screening mutant diapophytoene synthases, and/or a method for screening enzymes related to the carotenoid precursor supply pathway.

Claims (28)

A carotenoid represented by any one of the following formulas.
A carotenoid represented by the following formula:
A carotenoid represented by the following formula:
A carotenoid represented by the following formula:
A carotenoid represented by the following formula:
A carotenoid represented by the following formula:
A carotenoid represented by the following formula:
A carotenoid represented by the following formula:
A carotenoid represented by the following formula:
A carotenoid represented by the following formula:
A carotenoid represented by the following formula:
A method for producing a carotenoid having an X chain and a Y chain and having five conjugated double bond structures, comprising the steps of:
A)
1) a step of desaturating a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain of a carotenoid having an X chain and a Y chain and having three conjugated double bond structures present at the junction between the X chain and the Y chain;
Or,
B)
1) A step of producing a carotenoid having an X chain and a Y chain and having three conjugated double bond structures at the connecting portion between the X chain and the Y chain by condensing two molecules of a prenyl diphosphate having n isoprenoid units and a prenyl diphosphate having m isoprenoid units;
2) a step of desaturating a single bond adjacent to three conjugated double bonds present at the connection between the X chain and the Y chain of the carotenoid produced in 1);
wherein X is a structure in which n isoprenoid units (C5) are bonded in a linear chain, Y is a structure in which m isoprenoid units (C5) are bonded in a linear chain, the terminal carbon of the X chain forms a double bond with the terminal carbon of the Y chain to form three conjugated double bond structures, and n and m are integers of 1 to 8, but are different from each other. The method according to claim 12, wherein the desaturation step is a step of reacting an enzyme that desaturates a single bond adjacent to the three conjugated double bond structures present at the connection between the X chain and the Y chain. The method of claim 13, wherein the enzyme substantially desaturates only the single bonds on the X-chain side adjacent to the three conjugated double bond structures. The method of claim 12, wherein the X chain has 15 carbon atoms, the Y chain has 10 carbon atoms, and only the single bonds in the X chain adjacent to the three conjugated double bond structures are substantially unsaturated. The method of claim 12, wherein the X chain has 15 carbon atoms, the Y chain has 5 carbon atoms, and only the single bonds in the X chain adjacent to the three conjugated double bond structures are substantially unsaturated. The method of claim 12, wherein the X chain has 20 carbon atoms, the Y chain has 25 carbon atoms, and only the single bonds in the X chain adjacent to the three conjugated double bond structures are substantially unsaturated. The method of claim 12, wherein the X chain has 20 carbon atoms, the Y chain has 30 carbon atoms, and only the single bonds in the X chain adjacent to the three conjugated double bond structures are substantially unsaturated. The method of claim 12, wherein the X chain has 15 carbon atoms, the Y chain has 30 carbon atoms, and only the single bonds in the X chain adjacent to the three conjugated double bond structures are substantially unsaturated. The method of claim 12, wherein the X chain has 20 carbon atoms, the Y chain has 15 carbon atoms, and only the single bonds in the X chain adjacent to the three conjugated double bond structures are substantially unsaturated. The method of claim 12, wherein the X chain has 15 carbon atoms, the Y chain has 25 carbon atoms, and only the single bonds in the X chain adjacent to the three conjugated double bond structures are substantially unsaturated. The method according to any one of claims 12 to 21, wherein the carotenoid having an X chain and a Y chain and having five conjugated double bond structures is a fluorescent carotenoid. The method according to claim 12, wherein the method is carried out in a cell into which any one of the following genes (1) to (4) has been introduced or which is endogenous to the cell:
(1) a prenyl diphosphate synthesis gene having n isoprenoid units; (2) a prenyl diphosphate synthesis gene having m isoprenoid units; (3) an enzyme gene that condenses two molecules of a prenyl diphosphate having n isoprenoid units and a prenyl diphosphate having m isoprenoid units; (4) an enzyme gene that desaturates a single bond adjacent to three conjugated double bond structures present at the junction between the X chain and the Y chain;
The method for producing the carotenoid is characterized in that it is carried out by culturing the cells in a medium containing an organic solvent while extracting the carotenoid having an X chain and a Y chain and having five conjugated double bond structures from the solvent. A method for screening mutant diapophytoene synthases having the activity of bicondensing two different sizes of prenyl diphosphate, comprising the steps of: introducing a gene library of diapophytoene synthases (CrtM) into cells capable of synthesizing a carotenoid having an X chain and a Y chain and five conjugated double bond structures from a carotenoid having an X chain and a Y chain and three conjugated double bond structures; and selecting the cells using the fluorescence intensity of the cells as an index. The method for screening a mutant diapophytoene synthase having the activity of bicondensing two prenyl diphosphate molecules of different sizes according to claim 24, wherein the X chain has 15 carbon atoms, the Y chain has 10 carbon atoms, and the single bonds adjacent to the three conjugated double bond structures in the X chain are desaturated. A mutant diapophytoene synthase (CrtM) in which one or more mutations selected from L160W and A245T have been introduced into the wild type and which has the activity of condensing two molecules of prenyl diphosphate of different sizes. 27. The mutant diapophytoene synthase (CrtM) of claim 26, in which the mutations L160W, E180G, and A245T are introduced into the wild type. The mutant diapophytoene synthase (CrtM) according to claim 27, further comprising a G138A or G138S mutation.

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